Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Plasmids are an integral part of the horizontal gene pool and, therefore, are the main
vectors for the spread of antibiotic and heavy metal resistance genes in the
environment. Functional and taxonomic characterization of novel plasmids is, therefore,
central to our general understanding of plasmid biology and their contribution to
microbial evolution. Two 14-kb mobilizable plasmids, p31T1 and p36T2, conferring
resistance to tetracycline were isolated from the opportunistic fish pathogens
Aeromonas sobria and Aeromonas hydrophila and were found to have indistinguishable
restriction fragment length polymorphism (RFLP) patterns (Marx, MSc Thesis). DNA
sequence analysis of the two isogenic plasmids (only p36T2 was sequenced) revealed
the presence of 18 putative open reading frames (ORFs), of which the tetAR
tetracycline resistance genes, associated with a truncated Tn1721, were the only ORFs
with significant similarity to known sequences within the NCBI database. Putative
functions were assigned to 10 of the ORFs based on their distant homology with
proteins of known function. Six of the 18 ORFs, spanning 5.7-kb, were found to
comprise the minimal region required for replication (minimal replicon) by means of
deletion analysis using derivatives of p31T1. Of the six ORFs, ORF2 and ORF4 were
found to be essential for plasmid replication. Inactivation of ORF3 resulted in an
increase of plasmid copy number (PCN) from ~3 to ~7 plasmids per chromosome and a
decrease in plasmid stability from ~80 % to 16 % over approximately 127 generations (7
days). Furthermore, by means of β-galactosidase promoter fusion assays it was shown
that ORF3 autoregulated its own promoter. These results, therefore, suggested that
although ORF3 was not essential for replication, it may be involved in plasmid copy
number regulation and control. Host range analysis indicated that p31T1 was able to
replicate in two other members of the γ-proteobacteria group (Escherichia coli and
Pseudomonas putida) but was unable to do so in an α-proteobacterium strain, thus
suggesting a limited host range. Furthermore, p31T1 was mobilized only at low
frequencies (5.4 x 10-5 transconjugants per donor) by an IncP-1 conjugative system
though it is possible that the mobilization system of these plasmids is adapted to function optimally with alternate conjugative systems. Given the unique PCN, stability,
host range and mobilization characteristics determined for p31T1 and that no other
plasmid replication and mobilization systems with significant sequence similarity to
these plasmids have yet been identified, it is likely that these two plasmids are the first
representative members of a new family of plasmids found within aquacultureassociated
Aeromonas species and which are involved in the spread of tetracycline
resistance. / AFRIKAANSE OPSOMMING: Plasmiede vorm ‘n integrale deel van die horisontale geen poel en vorm daarom die
hoof vektore vir die verspreiding van antibiotika- en swaarmetaal-weerstandbiedende
gene in die omgewing. Funksionele en taksonomiese karakterisering van nuwe
plasmiede is belangrik in die begrip van plasmied biologie en hul bydrae tot mikrobiese
evolusie. Twee 14-kb mobiliseerbare plasmiedes, p31T1 en p36T2, met tetrasiklien
weerstandigheid was vanaf die opportunistiese vis patogene Aeromonas sobria en
Aeromonas hydrophila geïsoleer en het identiese restriksie fragment lengte
polimorfisme (RFLP) patrone. DNA volgorde analise van die twee isogeniese plasmiede
(slegs die volgorde van p36T2 was bepaal) het die teenwoordigheid van 18 moontlike
oop leesrame (OLR) getoon. Die tetAR tetrasiklien weerstandbiedende gene, wat met ‘n
verkorte Tn1721 transposon geassosieerd is, was die enigste OLR wat beduidende
volgorde ooreenkoms met bekende volgordes binne die NCBI databasis getoon het.
Moontlike funksies was toegeken aan 10 van die OLRe en was gebasseer op vêrlangse
homologie met proteïene met bekende funksies. Ses van die 18 OLRe strek oor ‘n 5.7-
kb minimale replikon fragment wat benodig word vir replisering en is deur middel van
delesie analises van p31T1 derivate gevind. Van hierdie ses OLRe, word OLR2 en
OLR4 benodig vir plasmied replisering. Inaktivering van OLR3 het ‘n toename in
plasmied kopiegetal (PKG) vanaf ~3 tot ~7 plasmiede per kromosoom en ‘n afname in
stabiliteit vanaf ~80% tot 16% oor 127 generasies (7 dae) tot gevolg gehad. Verder kon
daar deur middel van β-galaktosidase fusie analises getoon word dat OLR3 sy eie
promotor outoreguleer. Hierdie resultate stel dus voor dat alhoewel OLR3 nie benodig
was vir replikasie nie, mag dit dalk by plasmied kopiegetal regulering en beheer
betrokke wees. Bakteriële gasheer analises het getoon dat p31T1 in 2 addisionele lede
van die γ-proteobakterieë groep (Escherichia coli en Pseudomonas putida) kon
repliseer, maar nie in ‘n α-proteobacterium nie. Verder kon p31T1 teen ‘n lae frekwensie
(5.4 x 105) gemobiliseer word deur ‘n IncP-1 konjugasie sisteem, maar dit mag wees dat
die mobilisering eerder optimaal kan plaasvind met ‘n alternatiewe konjugasie sisteem.
Na aanleiding van die unieke PKG, stabiliteit, gasheer en mobilisering eienskappe wat vir p31T1 bepaal is en die feit dat geen ander replisering en mobilisering sisteme met
noemenswaardige volgorde homologie tot hierdie plasmiede gevind kon word nie, blyk
dit dat hierdie van die eerste lede van ‘n nuwe familie van plasmiede binne die
akwakultuur-geassosieerde Aeromonas spesies is, wat betrokke is by die verspreiding
van tetrasiklien weerstandbiedendheid.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/85725 |
| Date | 12 1900 |
| Creators | Laubscher, Inge |
| Contributors | Rawlings, D. E., Deane, S. M., Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | viii, 151 p. : ill. |
| Rights | Stellenbosch University |
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