Lactic acid bacteria (LAB), a group of generally recognized as safe (GRAS) organisms that metabolize sugars into primarily lactic acid, have traditionally been used for the fermentation and preservation of various foods and beverages. There is increasing interest in the genetic manipulation of LAB to improve existing characteristics or introduce novel, industrially pertinent phenotypes. However, because these bacteria have food-related applications, their genetic modification requires the use of food-grade genetic engineering tools. LAB plasmids, self-replicating extrachromosomal DNA molecules, can be used to derive food-grade cloning vectors. The rationale of this research was to develop a food-grade cloning vector using a lactobacilli cryptic plasmid and to investigate its cloning and expression properties. The main objectives were to (i) screen Lactobacillus spp. for plasmids, (ii) isolate and characterize a plasmid, and (iii) use the plasmid replicon to construct a cloning vector and express heterologous genes in various hosts. This is the first step in the development of a new family of food-grade cloning vectors for the genetic modification of lactobacilli.

Three overlapping DNA fragments from the lower operon of Pseudomonas putida TOL plasmid pDK1, covering the xy1IH genes and downstream flanking region, were cloned into pUC19. They include a 2.8 kbp XhoI fragment, a 2.7 kbp PstI fragment and a 2.0 kbp EcoRI-HindIII fragment. They were subjected to DNA sequence analysis. The xy1I (4-oxalocrotonate decarboxylase) and xy1H (4-oxalocrotonate tautomerase) genes were found to possess coding regions of 792 and 189 nucleotides, respectively. A possible transcriptional terminator resembling E. coli rho-independent terminators was identified downstream of the translational stop of xy1H. An additional stem and loop structure was found in the intergenic region between xy1I and xy1H. The individual ORF's of the oxalocrotonate branch (xy1G, xy1I and xy1H) have been cloned into pUC18/19. The expression of the xy1G gene in E. coli was successfully assayed spectrophotometrically.

A pyrA mutation in Pseudomonas putida was isolated using transposon mutagenesis for the first time. Transposon Tn5 was used to inactivate the pyrA gene for carbamoylphosphate synthetase in these mutants. Accordingly, these mutants were defective in pyrimidine and arginine biosynthesis. The suicide vector, pM075, from Pseudomonas aeruginosa, was used to introduce the transposon into the cells. Tn5 was subsequently used to supply homology so that the plasmid pM075 could be introduced in its entirety into the Pseudomonas putida chromosome at the locus of the Tn5 insertion in the pyrA gene. Consequently, these strains exhibited high frequency of recombination and were capable of chromosome mobilization.

Genetic engineering tools are under continuous development. However, hesitation by consumers and governments regarding consumption of genetically modified organism (GMO) affects taking advantage of developments in biotechnology. While being a complicated issue to address, this challenge inspired us to investigate whether it is possible to engineer organisms without altering their wild-type genomes, but with the same customizability level offered by genetic engineering; that is, having the capacity of expressing foreign proteins not codified by the wild-type genome. I used B. subtilis spores as a model organism for this purpose. I took advantage of the sporulation process during which two compartments with differential expression, or different gene expression patterns co-exist, the mother cell and the forespore, and I programmed a single designer plasmid to behave differently in each compartment: the plasmid in the mother cell modifies the spore phenotype, while the plasmid in the forespore undergoes self-digestion. At the end of sporulation, the mother cell lyses and releases the final product — a plasmid-free engineered spore. Following this, I incorporated the forespore-specific "self-digestion" gene circuit into a variety of plasmids with different purposes, including the generation of spores expressing GFP on their protective coats and the artificial induction of sporulation, both of them as a proof-of-concept of genetically intact bioengineered organisms. Production of the different types of genetically intact bioengineered spores resulted in an average of nearly 90% of them free of detectible plasmid or genome alterations. Spores of B. subtilis and other species overall continue to gain attention in the biotechnology sector, with potential applications ranging from biopesticides, probiotics, and vaccines to energy-converting materials, self-healing concrete, and whole-cell biocatalysts. While spores represent a special case of multiple-compartment organisms among bacteria, most eukaryotic organisms possess multiple compartments, structures, or tissues with differential expression, including plants and animals. Therefore, our results in this study could serve as a starting point for new ideas and methods for the genetic modification-free engineering of complex organisms or parts of them.

No description available.

Thesis (MSc (Microbiology))--University of Stellenbosch, 2005. / Plasmid pTC-F14 is a 14.2kb promiscuous, broad-host range IncQ-like mobilizable plasmid isolated from Acidithiobacillus caldus f. At. caldus is a member of a consortium of bacteria (along with Acidithiobacillus ferrooxidans and Leptospirilum ferrooxidans) that is used industrially for decomposing metal sulphide ores and concentrates at temperatures of 40ºC or below which is now a well-established industrial process to recover metals from certain copper, uranium and gold-bearing minerals or mineral concentrates. These biomining microbes are usually obligately acidophilic, autotrophic, usually aerobic iron- or sulphur-oxidizing chemolithotrophic bacteria. Their remarkable physiology allows them to inhabit an ecological niche that is largely inorganic and differs from those environments populated by the more commonly studied non-acidophilic heterotrophic bacteria. At. caldus, is a moderately thermophilic (45 to 50ºC), highly acidophilic (pH1.5 to 2.5) sulphur-oxidizing bacterium, and its role as one of the major players in the industrial decomposition of metal sulphide ores has become evident in recent years. At. caldus f from which pTC-F14 was isolated was found to be one of two dominant organisms in a bacterial consortium undergoing pilot-scale testing for the commercial extraction of nickel from ores.

Thesis (MScMedSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: The plasmid pAL5000 is a mycobacterial plasmid isolated from Mycobacterium fortuitum. It is a low copy number plasmid, which replicates in both fast growing (e.g. M. smegmatis) and slow growing (e.g. M. bovis BCG) mycobacteria. Most mycobacterial-E. coli shuttle vectors utilise the pAL5000 origin of replication. The minimum replicon consists of ORF1 (RepA), ORF2 (RepB) and the origin of replication. Dr W.R. Bourn created an E. coli-mycobacterial vector based on the pAL5000 origin of replication (pORI) and then subjected it to semi-random mutagenesis. A high copy number mutant was identified (pHIGH) and the causative mutation was tentatively identified as a 3bp deletion situated just upstream of repB. This work describes the further characterisation of the mutant plasmid. Firstly, it was shown by retransforming M. smegmatis with both the original and mutant plasmids (pORI and pHIGH), that the mutation causing the increased copy number was plasmid-encoded and not on the chromosome. Following this, it was demonstrated by simple subcloning of the region that carries the 3 bp deletion, that other pAL5000-based vectors could be converted to high copy number. In addition to this, the subcloned region was sequenced and the nature of the mutations was confirmed. The subcloning experiment confirmed that the 3bp deletion caused the high copy number phenotype. Following this, the exact copy number of pHIGH and the relative increase in copy number was determined. From this, the copy number of pORI could also be determined. The plasmid pHIGH has a copy number of approximately 54, compared to the 8 of pORI (a relative increase by a factor of 7). Because it is important for researchers to know the characteristics of the vectors that they use, especially the influence it will have on its host, stability tests and growth curves were also performed. It was seen that the higher copy number did not markedly increase the stability, however, this is because pORI is already extremely, and unexpectedly, stable in the host M. smegmatis. According to the growth curves, the increased copy number has little effect on the growth of the host M. smegmatis. Possible mechanisms for the increased copy number were then investigated. By using a promoter probe vector, the possible existence of a promoter situated between the two open reading frames of pAL5000 (repA and repB) was investigated. It was thought that the mutation might have created, or changed an existing promoter, situated between repA and repB. The results showed, however, that in both pORI and pHIGH there might be a very weak promoter upstream of repB, but the mutation did not cause any change that was measurable by the method that was used. A further possibility was that the mutation caused a change in the RNA secondary structure, which might then have an effect on the translational efficiency of RepB. It was found that the 3bp deletion in pHIGH causes a change in the local RNA secondary structure around the ribosomal binding site and the start codon, when compared to pORI (wild type). This change may cause the translation initiation rate of RepB to be different between pHIGH and pORI. Ultimately it would lead to a different ratio of RepA and RepB in the cell. / AFRIKAANSE OPSOMMING: Die plasmied pAL5000 is ‘n mikobakteriele plasmied wat vanuit Mycobacterium fortuitum gei'soleer is. Dit is ‘n lae kopie-getal plasmied wat in beide vinnig groeiende (bv. M. smegmatis) en stadig groeiende (bv. M. bovis BCG) mikobakteriee kan repliseer. Die meeste mikobakteriele-E. coli shuttle vektore gebruik die pAL5000 oorsprong van replisering. Die minimum replikon bestaan uit ORF1 (RepA), ORF2 (RepB) en die oorsprong van replisering. Dr. W.R. Bourn het ‘n E. coli-mikobakteriele vektor gemaak wat gebaseer is op die pAL5000 oorsprong van replisering (pORI), en dit onderwerp aan semi-random mutagenese. ‘n Hoë kopie-getal mutant is gei'dentifiseer (pHIGH) en die mutasie hiervoor verantwoordelik was tentatief gei'dentifiseer as ‘n 3bp delesie, net stroomop van repB. Die projek beskryf die verdere karakterisering van die mutante plasmied. Eerstens, deur M. smegmatis te hertransformeer met die plasmied DNA (pORI en pHIGH), is dit bewys dat dit mutasie wat die toename in kopie-getal veroorsaak, deur die plasmied gekodeer word, en dat dit nie ‘n mutasie op die chromosoom is nie. Hierna is dit deur eenvoudige subklonering bewys dat die gedeelte wat die 3bp delesie dra, ander pAL5000-gebaseerde vektore ook kan verander in ‘n hoër kopie-getal. Die sub-klonerings eksperiment het ook bewys dat die 3 bp delesie die oorsaak is vir die hoë kopie-getal fenotipe. Volgende is die presiese kopie-getal van pHIGH en die relatiewe toename in kopiegetal bepaal. Die kopie-getal van pORI kon vanaf hierdie data bepaal word. Die plasmied pHIGH het ‘n kopie-getal van ongeveer 54 in M. smegmatis, in vergelyking met die 8 van pORI (‘n relatiewe toename met ‘n faktor van 7). Aangesien dit vir navorsers belangrik is om die eienskappe van die vektore wat hulle gebruik, te ken, en veral die invloed wat dit op die gasheer sal hê, is stabiliteits toetse, en groeikurwes gedoen. Die hoër kopie-getal het nie die stabiliteit werklik verbeter nie, maar dit is omdat pORI alreeds uiters stabiel is in die gasheer M. smegmatis. Volgens die groeikurwes het die toename in kopie-getal ‘n minimale effek op die groei van die gasheer M. smegmatis. Moontlike meganismes vir die hoër kopie-getal is ook ondersoek. Die moontlike bestaan van ‘n promoter tussen die twee oop-leesrame van pAL5000 (repA en repB) is ondersoek deur gebruik te maak van ‘n “promoter probe” vektor. Die mutasie kon moontlik ‘n promoter geskep het, of ‘n bestaande een tussen repA en repB verander het. Die resultate het gewys dat daar in beide pORI en pHIGH moontlik ‘n baie swak promoter stroomop van repB is, maar die mutasie het nie enige veranderinge veroorsaak wat meetbaar was met die metode wat gebruik is nie. ‘n Verdere moontlikheid was dat die mutasie ‘n verandering in die RNA sekondere struktuur kon veroorsaak het, en dit mag ‘n effek hê op die translasie effektiwiteit van RepB. Daar is gevind dat, in vergelyking met pORI, het die 3bp delesie in pHIGH ‘n verandering in die lokale RNA sekondere struktuur rondom die ribosomale bindings posisie en die begin-kodon veroorsaak. Die verandering mag veroorsaak dat die translasie inisiasie tempo van RepB verskillend is vir pORI en pHIGH. Uiteindelik sal dit lei tot ‘n heeltemal ander verhouding van RepA en RepB in die sel.

The TOL plasmids of Pseudomonas putida encode enzymes required for the oxidation of toluene and other related aromatic compounds. These genes are organized into two operons, the xylUWCMABN operon (upper), and the xylXYZLTEGFJQKIH operon (lower). Here we report the nucleotide sequence of a 7107 bp segment of the TOL pDK1 plasmid encoding the region just upstream of the "upper" operon through the genes encoding xylUWCMA. Sequence analysis, comparison of base-usage patterns, codon-usage patterns, and intergenic distances between genes help support the idea that the "upper" and "lower" operons have evolved independently in different genetic backgrounds and have only more recently been brought together in TOL and related catabolic plasmids.

Bibliography: leaves 104-130. The aim of this thesis is to characterise subgenomic RNAs of cucumoviruses and the functions of their encoding genes. Strains of cucumber mosaic virus (CMV) are classified into two major subgroups (I and II) on the basis of nucleotide sequence homology. The V strain of tomato aspermy virus (V-TAV) and a subgroup I CMV strain (WAII) are chosen to determine whether the 2b genes encoded by these viruses are expressed 'in vivo'. For further investigation of the 2b gene function, cDNA clones of three genomic RNAs of V-TAV are constructed. Using the infectious cDNA clones of V-TAV, a mutant virus containing only one of the two repeats is constructed.

Bibliography: leaves 104-130. / vi, 130, [25] leaves, [13] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The aim of this thesis is to characterise subgenomic RNAs of cucumoviruses and the functions of their encoding genes. Strains of cucumber mosaic virus (CMV) are classified into two major subgroups (I and II) on the basis of nucleotide sequence homology. The V strain of tomato aspermy virus (V-TAV) and a subgroup I CMV strain (WAII) are chosen to determine whether the 2b genes encoded by these viruses are expressed 'in vivo'. For further investigation of the 2b gene function, cDNA clones of three genomic RNAs of V-TAV are constructed. Using the infectious cDNA clones of V-TAV, a mutant virus containing only one of the two repeats is constructed. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997