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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.

Thesis (DSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Plasmids belonging to the IncQ family have an exceptionally broad host-range and are highly mobilizable in the presence of the self-transmissible IncP plasmids. All IncQ plasmids identified to date have certain features in common. The feature that distinguishes them most from all other plasmids is that they have a unique mechanism of replication. Their replicons consist of repA, repB and repC genes encoding a replicase, primase and DNA-binding proteins respectively. All IncQ plasmids contain at least three 22-bp iterons (or 20-bp iterons with 2-bp spacers) that are identical in sequence and to which the RepC DNA-binding protein binds. They replicate by means of a unique strand-displacement mechanism that is considered to place a limit on their size. Replication proceeds by a partially single-stranded intermediate that is believed to result in an increased likelihood of structural instability with an increase in plasmid size. The most compact backbone of IncQ plasmids is approximately 5.9-kb and the largest natural IncQ plasmid reported is 14.2-kb. Although the mobilization regions of IncQ plasmids are not as unique as the replicons, they are all characterized by the primase of the replicon being fused to the relaxase of the mobilization genes. The remainder of the mobilization genes may vary substantially in number and sequence between plasmids and have been subdivided into at least four distinct lineages. This dissertation consists of twenty one manuscripts published during the period 1984 to 2012. The focus is almost entirely on the IncQ plasmid subfamily known as IncQ2. Most of the earlier work was on determining the nature and extent of the replicons, mobilization genes and the toxin-antitoxin plasmid stability system. A strong theme in the latter work focussed on using the natural variation among the IncQ2 plasmids as a means to understand IncQ plasmid evolution. The collection of articles comprises a combination of original research and reviews. / AFRIKAANSE OPSOMMING: Plasmiede wat aan die IncQ familie behoort kom ‘n uitsonderlike wye gasheerselreeks voor en is hoogs mobiliseerbaar deur middel van die selfoordraagbaar IncP plasmiede. Alle IncQ plasmiedes wat tot datum identifiseer is het sekere gemeenskaplike eienskappe. Die eienskap wat hulle van alle ander plasmiedes onderskei is hul unieke dupliseringsmeganisme. Hul dupliseringsmeganisme bestaan uit repA, repB en repC gene wat onderskeidlik ‘n helikase, ‘n ‘primase’ en ‘n DNSbindingsproteïen enkodeer. Die IncQ plasmiede het ten minste drie 22-bp iterone (of 20-bp iterone met 2-bp skeidingsnukleotiede) met ‘n identiese nukleotiedvolgorde en waaraan die RepCbindingsproteïen bind. Hulle dupliseer deur middel van ‘n unieke DNA-string-vervangingsmeganisme wat ‘n beperking op hul grootte plaas. Tydens replikasie word ‘n intermediêre struktuur gevorm wat gedeeltelik enkelstring is en dit is blykbaar die rede vir ‘n verhoging in strukturële onstabilitiet as die plasmied groter word. Die kleinste ruggraat onder die IncQ plasmiede is min of meer 5.9-kb en die grootste natuurlike IncQ plasmied wat gerapporteer is, is 14.2-kb. Alhoewel die mobiliseringsgebied van die IncQ plasmiede nie so duidelik uitkenbaar as die replikons nie, hierdie gebied is gekenmerk deur ‘n ‘primase’ wat aan ‘n ‘relaxase’ in die mobiliseringsgene gekoppel is. Die oorblywende mobiliseringsgene verskil in beide getal en nukleotiedvolgorde tussen plasmiede en is gebruik om die plasmiede in vier duidelike oorsponggroepe in te deel. Hierdie proefskrif bestaan uit een-en-twintig artikels wat tussen 1984 en 2012 gepubliseer is. Die fokus is hoofsaaklik op die IncQ plasmiedsubfamilie wat as IncQ2 bekend is. Baie van die vroeër werk het oor die aard en omvang van die duplisering en mobiliseringsgene asook die toksienteentoksien plasmiedstabiliseringsmeganisme hanteer. ‘n Sterk tema in die latere werk was om die natuurlike variasie onder die IncQ2 plasmiede te bestudeer ten einde IncQ plasmiedevolusie te verstaan. Die publikasie versameling bestaan uit ‘n kombinasie van oorspronklike navorsing en oorsigartikels.

Enterococcus faecalis are Gram-positive bacteria that colonize the gastrointestinal tracts of mammals, birds and invertebrates and are also found in sewage, soil, food and water. In addition to being commensal organisms, Enterococci can also cause nosocomial infections in humans including urinary tract infections, septicemia and endocarditis. Hospital-acquired infections often present a challenge in treatment due to the emergence of multi-drug resistant strains. Enterococcal plasmids may act as extremely stable reservoirs for resistance genes and other virulence factors. Pheromone responsive plasmids such as pCF10 mediate efficient transfer of genetic material within the species E. faecalis but may also be capable of transferring resistance genes across species and genus boundaries. Polymicrobial environments often found in nosocomial infections may expose plasmid-harboring enterococci to pathogenic species, poising cells for this type of promiscuous horizontal gene transfer of resistance determinants. Previous studies showed that prgW, which encodes the pCF10 replication initiation protein PrgW, is the minimal origin of replication for this plasmid. The replicon, which is usually limited to Enterococcal spp., can replicate in Lactococcus lactis if it is engineered to produce pre-cCF10. Three conserved cysteines (C78/C275/C307) are important for plasmid stability and allow for replication of the pCF10 replicon in L. lactis in the absence of pre-cCF10. PrgW has a predicted molecular weight of 38,635. Four polyclonal antibodies targeting PrgW at the N-terminus (aa 1-20), C-terminus (aa 314-333) and two internal regions (aa 64-80 and aa 250-271) were used in current experiments and retrospective studies. When PrgW was overexpressed in E. faecalis, four different apparent approximate molecular weights were detected by Western blotting (p40*, p36*, p24* and p18*), suggestive of processing. In Enterococci where the replicon is active, p36* was consistently detected by all four antisera; when PrgW was overexpressed in Streptococcus mutans where the replicon is non-functional, p49* and p40* were detected but p36* was not observed. PrgW p24* was detected by a mixture of the internally targeting antibodies as well as the C-terminal targeting antibody, but not the N-terminal targeting antibody, suggesting that the N-terminal domain of PrgW has been cleaved off in p24*. The p24* form may play a role in pCF10 stability. Mutations to three cysteines in PrgW (C78/C275/C307), which reduce the stability of pCF10, result in the loss of p24*. Enterococcal conjugative plasmids have been previously implicated in the transfer of antibiotic resistance genes. The pCF10 plasmid contains the conjugative transposon Tn925, which possesses the tetM tetracycline resistance gene. Proximity of donor and recipient cells is a key part of pheromone-responsive conjugation. Aggregation substance allows for formation of clumps of E. faecalis in liquid mating experiments. E. faecalis forms biofilms; in contrast to filter mating experiments, polymicrobial biofilms provide an in vitro model of a natural scenario during which horizontal gene transfer may occur. Rates of cross-genus genetic transfer of tetM between E. faecalis OG1RF(pCF10) donor cells and Staphylococcus aureus recipient cells growing on glass coverslips as mixed-species biofilm populations were determined to be 10-8 after pheromone induction of pCF10 conjugation. This biofilm transfer model also holds potential to test the efficacy of synthetic peptides in the reduction or even prevention of pCF10 transfer, and the consequential dissemination of antibiotic resistance determinants throughout the genus Enterococcus and beyond. / Microbiology and Immunology