Thesis (PhD (Microbiology))--University of Stellenbosch 2010. / ENGLISH ABSTRACT: Two isogenic plasmids, pRAS3.1 (11,851-bp) and pRAS3.2 (11,823-bp), were identified as tetracycline resistance plasmids occurring within Aeromonas salmonicida subsp. salmonicida and atypical A. salmonicida subsp. salmonicida strains that were isolated from salmon aquaculture farms in Norway (L'Abee-Lund and Sorum, 2002). Although sequence analysis showed that, except for the repC gene, the replication and mobilization genes of the two pRAS3 plasmids are similar to that of the two IncQ-2 plasmids pTF-FC2 and pTC-F14, incompatibility testing during the course of this study revealed that the replicons of the two pRAS3 plasmids were compatible with the replicons of the IncQ-1α, ß] and y plasmids RSF1010, pIE1107 and pIE1130, as well as with the IncQ-2α and ß plasmids, pTF-FC2 and pTC-F14, respectively. Through sequence analysis it was suggested that the repC gene of the ancestral pRAS3 plasmid was probably acquired during a gene exchange event with a yet to be identified plasmid. The difference in the RepC of the pRAS3 plasmids compared to that of the other IncQ-like plasmids against which the pRAS3 plasmids were tested for incompatibility was thus suggested to be a likely reason for the compatibility of the two pRAS3 plasmid replicons with these IncQ-1 and IncQ-2 plasmids. Two previously unidentified genes, encoding two small 108 and 74-aa proteins distantly related to the PemIK (Bravo et al., 1987; Tsuchimoto et al., 1988) and MazEF (Masuda et al., 1993) TA systems, were found to be present between repB and repA genes of the two pRAS3 plasmids. Cloning of these two genes onto an unstable pOU82-test vector increased the stability of the vector from 35 to 98% after ~72 generations, thus suggesting that like the PasABC and PasAB systems of pTF-FC2 and pTC-F14, these two genes encode proteins which function as a toxin-antitoxin (TA) system. Although located in a similar position on the plasmids, the TA system of the two pRAS3 plasmids and the Pas systems of pTF-FC2 and pTC-F14 are unrelated, suggesting that these two types of TA systems were acquired independently from each other. Based on the sequence similarity and genetic organization of pRAS3 compared to the IncQ-2α and ß] plasmids pTF-FC2 and pTC-F14, respectively, but given that the pRAS3 plasmids were compatible with both pTF-FC2 and pTC-F14, as well as other IncQ-like plasmids, it was suggested that the two pRAS3 plasmids be classified into a new IncQ-2y subgroup. A comparison of the sequences of the two pRAS3 plasmids to each other by L'Abee-Lund and Sorum (2002) revealed that, apart from a number of point mutations within the tetAR tetracycline resistance genes of the two plasmids, the only other differences between them are that pRAS3.1 has 4 tandem copies of 22-bp iteron repeats within its origin of vegetative replication (oriV), and 5 tandem copies of CCCCCG 6-bp repeats near the origin of transfer (oriT), while pRAS3.2 has only three and four copies of each of the two repeated sequences, respectively. As the two pRAS3 plasmids are likely to have arisen from the same ancestor, this raised the question of how the copy numbers of these two different types of repeat sequences affected the ability of pRAS3.1 and pRAS3.2 plasmids to compete within a host cell as well as within a population of host cells, and therefore, why both of these isogenic plasmids have managed to persist in the environment. The plasmid copy numbers (PCN) of pRAS3.1 and pRAS3.2 were estimated to be 45 ± 13 and 30 ± 5 plasmids per chromosome, respectively. By creating a series of pRAS3.1 derivative plasmids with 3 to 7 copies of the 22-bp iterons and 4 or 5 copies of the 6-bp repeats, it was shown that an increase in the number of iterons brought about a decrease in PCN, probably due to an increased ability to bind RepC, while an increase in the number of 6-bp repeats from 4 to 5 brought about an increase in repB transcription, and the higher levels of RepB resulted in an increase in PCN. Thus the reason for pRAS3.1 having a ~1.5-fold higher PCN than pRAS3.2, even though it has 4 x 22-bp iterons compared to the 3 x 22-bp iterons of pRAS3.2, was that it had a higher level of repB transcription due to having 5 x 6-bp repeats in its mobB-mobA/repB promoter region compared to the 4 x 6-bp repeats of pRAS3.2. The differences in the number of iterons and 6-bp repeats, and hence PCN, did not have an effect on the stability of the two wild type (WT) plasmids or their derivatives even when the TA system was neutralized by having a copy of the TA genes present on a vector in trans and it was argued that the relatively high PCN of the two pRAS3 plasmids was sufficient to ensure plasmid stability through random distribution. As the two pRAS3 plasmids were mobilized at similar frequencies difference in PCN and mobB-mobA/repB transcription did not seem to affect their mobilization frequency. When pRAS3.1 and pRAS3.2 were competed intracellularly as coresident plasmids, pRAS3.1 was able to displace pRAS3.2 from 98% of the host cells within ~20 generations. The displacement of pRAS3.2 by pRAS3.1 was found to be as a result of pRAS3.1 having 4 x 22-bp iterons, which enabled pRAS3.1 to titrate of the communal pool of available RepC initiator proteins. Plasmids with 5 or 7 x 22-bp iterons, were however less effective at displacing a plasmid with 3 iterons, and it was speculated that plasmids with more than 4 x 22-bp iterons within their oriV were less successful at initiating replication than was a plasmid with 3 iterons within its oriV. A direct correlation was found between the PCN of a pRAS3 plasmid and the metabolic burden it imposed on its host. Thus pRAS3.1, as a result of its ~1.5-fold higher PCN than pRAS3.2 placed a small but significantly higher (~2.8%) metabolic load on its host compared to pRAS3.2. It was concluded that pRAS3.1 had a competitive advantage over pRAS3.2 when these plasmids were coresident within a single host (as would have been when the two plasmids first diverged from each other) as it was able to displace pRAS3.2. However, as a result of pRAS3.2 having a lower PCN, it placed a smaller metabolic burden on an isogenic host and this resulted in pRAS3.2 having an advantage over pRAS3.1 at the population level. Sequence remnants of pRAS3.2 from horizontal gene transfer suggested that pRAS3.2 was the original pRAS3 plasmid and thus that pRAS3.1 evolved from pRAS3.2. As the pRAS3.1 derivative plasmids that were constructed during the course of this study are likely to have been intermediates in the evolution of pRAS3.1 from pRAS3.2, I was able to speculate on the stepwise evolution of pRAS3.1 from pRAS3.2 based on the characteristics of these plasmids, and thus, how both macro- and microevolutionary events have contributed to the evolution of these two plasmids. / AFRIKAANSE OPSOMMING: Die twee isogeniese plasmiede, pRAS3.1 en pRAS3.2, was geidentifiseer as tetrasiklien weerstandbiedende plasmiede wat in Aeromonas salmonicida subsp. salmonicida en nie-tipiese A. salmonicida voorkom (L'Abee-Lund and Sorum, 2002). DNS volgorde analise deur L'Abee-Lund en Sorum (2002) het gewys dat die gene verantwoordelik vir replisering (uitsluitend die repC) en mobililisering naverwant is aan die van twee IncQ-2 plasmiede, pTF-FC2 en pTC-F14. Eksperimente tydens hierdie studie het egter gewys dat die repliserende sisteme van die twee pRAS3 plasmiede versoenbaar is met die repliserende sisteme van die IncQ-1α, ß and y plasmiede RSF1010, pIE1107 en pIE1130, sowel as die IncQ-2α en ß plasmiede, pTF-FC2 and pTC-F14, onderskeidelik. Analise van die aminosuur volgorde van die pRAS3 RepC proteien het gedui daarop dat die proteien taamlik verskil van die RepC proteiene van die naverwante plasmiede pTF-FC2 en pTC-F14, sowel as die van die IncQ-1 tipe plasmiede, en daar was voorgestel dat die voorsaat pRAS3 plasmied moontlik die repC geen bekom het vanaf 'n ander, nog onbekende, plasmied deur middel van horisontale geen uitruiling. Die verskil in die RepC van die pRAS3 plasmiede teenoor die van die ander IncQ plasmiede waarteen hulle getoets was vir onversoenbaarheid, was waarskynlik die rede waarom die pRAS3 plasmiede versoenbaar was met die IncQ-1 en IncQ-2 plasmiede. DNS volgorde analise tydens hierdie studie het die teenwoordigheid van twee, vantevore ongeidentifiseerde, klein 108 en 74 aminosuur proteiene onthul wat ver langs verwant is aan die PemIK (Bravo et al., 1987; Tsuchimoto et al., 1988) en MazEF (Masuda et al., 1993) toksien-antitoksien sisteme. Die gene wat kodeer vir hierdie toksien-antitoksien proteine kom tussen die repB en die repA gene van die twee pRAS3 plasmiede voor. Klonering van die toksien-antitoksien gene van die pRAS3 plasmiede op 'n ander onstabiele plasmied het die stabiliteit van hierdie plasmied verhoog van 35 tot en met 98% na ~72 generasies. Hierdie experiment het dus bevestig dat, soos die PasABC en PasAB sisteme van pTF-FC2 en pTC-F14 onderskeidelik, die twee gene 'n toksien-antitoksien sisteem kodeer wat die stabiliteit van 'n plasmied binne 'n bakteriese populasie kan verbeter. Alhoewel die toksien-antitoksien gene van pRAS3 op 'n soortgelyke posisie op die pRAS3 plasmiede voorkom as wat die pasABC en pasAB gene op hulle onderskeidelike pTF-FC2 en pTC-F14 plasmiede voorkom, is hulle nie verwant nie en dus was dit voorgestel dat die twee tipe toksien-antitoksien sisteme onafhanklik van mekaar verkry is. Aangesien die DNS volgorde en genetiese rangskikking van pRAS3 teenoor die IncQ-2α en ß plasmiede pTF-FC2 en pTC-F14, onderskeidelik, soortgelyk is, asook die feit dat die pRAS3 plasmiede versoenbaar was met pTF-FC2 en pTC-F14, sowel as ander IncQ tipe plasmiede, word dit voorgestel dat die twee pRAS3 plasmiede in 'n nuwe IncQ-2y subgroep ingedeel word. 'n Vergelyking van die DNS volgorde van die twee pRAS3 plasmiede deur L'Abee-Lund and Sorum (2002) het gewys dat, behalwe vir 'n paar puntmutasies binne die tetAR tetrasiklien weerstandsgene, verskil die twee net in die opsig dat pRAS3.1 het 4 agtereenvolgende kopiee van 22-bp 'iteron' herhalings wat gelee is binne sy replikasie oorsprong en 5 kopiee van 'n CCCCCG 6-bp herhaling wat naby sy oorsprong van oordrag gelee is, terwyl pRAS3.2 net 3 en 4 kopiee het van elk van die onderskeie volgorde herhalings. Dus die bestaan van twee plasmiede met verskillende kopiegetalle van die twee verskillende tipe DNA volgorde herhalings, maar wat vermoedelik afkomstig is vanaf dieselfde stam plasmied, bring die volgende oorhoofse vrae aangaande die plasmiede na vore: hoe beinvloed die DNS volgorde herhalings die vermoe van die twee plasmiede om binne 'n enkele gasheersel te kompeteer vir die beskikbare plasmied repliserings masjinerie, en hoe beinvloed dit die plasmied-gasheersel verhouding en dus hulle vermoe om te kompeteer op die populasie vlak, en laastens, hoekom het beide weergawes van die plasmied bly voortbestaan in die omgewing? Die plasmied kopiegetalle van pRAS3.1 en pRAS3.2 was eksperimenteel beraam by ongeveer 45 ± 13 en 30 ± 5 plasmiede per chromosoom in E. coli, onderskeidelik. Deur 'n reeks van pRAS3.1 derivate te skep met 3 tot 7 'iteron' herhalings en 4 of 5 kopiee van die 6-bp herhalings was dit bewys dat 'n toename in die hoeveelheid 'iterons' 'n afname in die plasmied kopiegetal veroorsaak, vermoedelik deur 'n verbeterde vermoe om RepC te bind, terwyl 'n verhoging van 4 tot 5 kopiee van die 6-bp herhaling 'n afname in die kopiegetal te weeg gebring het. Die repB geen van 'n plasmied met 5 x 6-bp herhalings was ~2-voud hoer uitgedruk as die van 'n plasmied met 4 x 6-bp herhalings, en dit was verder bewys dat 'n verhoogde vlak van repB transkripsie vanaf 'n L-arabinose induseerbare promoter in trans van 'n pRAS3 plasmied met 4 x 6-bp herhalings het 'n ~2-voud verhoging in plasmied kopiegetal teweeg gebring. Die rede dat pRAS3.1 'n ~1.5-voud hoer plasmied kopiegetal gehad het as pRAS3.2, was as gevolg van 'n hoer vlak van repB uitdrukking weens die feit dat pRAS3.1 5 x 6-bp herhalings in die mobB-mobA/repB promoter area het terwyl pRAS3.2 net 4 van die 6-bp herhalings in dieslefde posisie het. Sou pRAS3.1 4 x 22-bp 'iterons' gehad het, maar saam met 4 x 6-bp herhalings soos pRAS3.2, dan sou die plasmied kopiegetal 23 ± 2 plasmiede per chromosoom gewees het. Die verskil in die hoeveelheid 'iterons' en 6-bp herhalings, en dus die plasmied kopiegetal, het nie 'n effek op die stabiliteit van die wilde tipe plasmiede of hulle derivate gehad nie, selfs al was die toksien-antitoksien sisteem geneutraliseer deurdat daar 'n kopie van die toksien-antitoksien sisteem op 'n ander plasmied in trans van die pRAS3 plasmiede en hul derivate geplaas was. Die relatiewe hoe plasmied kopiegetal van die pRAS3 plasmiede, wat moontlik hoog genoeg was om plasmied stabiliteit deur middel van toevallige uitdeling te verseker, was voorgestel as die rede vir die hoe mate van plasmied stabiliteit. Soortgelyke frekwensies van mobilisasie vir pRAS3.1 en pRAS3.2 (0.032 ± 0.014 en 0.021 ± 0.013 transkonjugate per donateur, onderskeidelik) was waargeneem. Dus het dit geblyk dat die verskil in uitdrukking van die mobB-mobA/repB operon, sowel as die plasmied kopiegetal van die twee pRAS3 plasmiede, nie die mobiliserings frekwensie beinvloed het nie. Intrasellulere kompetisie tussen pRAS3.1 en pRAS3.2 het gewys dat pRAS3.1 die vermoe gehad om binne ~20 generasies pRAS3.2 vanuit 98% van die gasheerselle te skop. Daar was gewys dat die teenwoordigheid van 4 x 22-bp 'iterons' in die oorsprong van replikasie van pRAS3.1 die rede was vir die vermoe van hierdie plasmied om pRAS3.2 uit te kompeteer binne die gasheersel, moontlik deurdat die 4 x 22-bp 'iterons' beter in staat was om die RepC protein te bind. Die vermoe van plasmiede met 5 of 7 x 22-bp 'iterons' om te kompeteer met 'n plasmied met net 3 x 22-bp 'iterons' was toenemend swakker in vergelyking met die van 'n plasmied met 4 x 22-bp 'iterons', en hierdie waarneming het gelei tot die voorstel dat plasmiede met meer as 4 x 22-bp 'iterons' nie so suksesvol was om replikasie te inisieer soos ¡¥n plasmied met 3 x 22-bp 'iterons' nie. 'n Direkte korrelasie was gevind tussen die plasmied kopiegetal van 'n pRAS3 plasmied en die metaboliese lading wat die plasmied op die gasheersel geplaas het. Dus het pRAS3.1, met 'n plasmied kopiegetal van ~1.5-voud hoer as die van pRAS3.2, 'n effens hoer (~2.8%) metababoliese lading op die gasheersel as pRAS3.2 geplaas. In gevolge van die inter- en intrasellulere kompetiesie eksperimente, was dit ge-argumenteer dat pRAS3.1 'n mededingende voordeel bo-oor pRAS3.2 binne 'n gasheersel (soos wat dit sou gewees het kort nadat die twee plasmiede van mekaar uiteengevloei het) gehad het omdat dit in staat was om pRAS3.2 vanuit die gasheersel te skop. Aan die ander kant het pRAS3.2 'n laer plasmied kopiegetal en dus 'n laer metaboliese lading op die isogeniese gasheersel geplaas het, en daardeur het pRAS3.2 weer op die populasievlak die kompeterende voordeel bo-oor pRAS3.1 gehad. Die eienskappe van pRAS3.2 was meer soortgelyk aan die van ander IncQ-tipe plasmiede as wat die eienskappe van pRAS3.1 was, en dus word dit voorgestel dat pRAS3.1 vanaf pRAS3.2 afkomstig was. Omdat die derivaat plasmiede wat geskep was vanaf pRAS3.1 tydens hierdie studie moontlike tussengangers in die ontwikkeling van pRAS3.1 vanaf pRAS3.2 was, kan gespekuleer word, gebaseer op die eienskappe van hierdie plasmiede, oor die “stapsgewyse manier” waarmee pRAS3.1 vanaf pRAS3.2 ge-evolueer het, en dus hoe beide makro- en mikro-evolusionere gebeurlikhede bygedra het tot die evolusie van genoemde plasmiede.

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.