Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The moderately thermophilic (45 to 50DC), highly acidophilic (pH 1.5 to 2.5),
chemolithoautotrophic Acidithiobaci/lus caldus strain "f' was isolated from a
biooxidation process used to treat nickel ore concentrates. Trans-Alternating Field
Electrophoresis (TAFE) analysis of total DNA from the At. caldus cells revealed two
plasmids of approximately 14 and 45-kb. The 14-kb plasmid, designated pTC-F14,
was cloned and shown by replacement of the cloning vector with a kanamycin
resistance gene to be capable of autonomous replication in Escherichia coli.
Autonomous replication was also demonstrated in Pseudomonas putida and
Agrobacterium tumefaciens LBA 4404 which suggested that pTC-F14 was a broad
host-range plasmid. Sequence analysis of the pTC-F14 replicon region revealed five
open-reading frames, and a replicon organization like that of the broad host-range
IncQ plasmids. Three of the open-reading frames encoded replication proteins with
amino acid sequence identities similar to that of the IncQ-like plasmid pTF-FC2
(RepA, 81%; RepB, 78%; RepC, 74%). This high level of relatedness suggested that
the two replicons had evolved from a common ancestor. Since closely related
replicons are usually incompatible, the compatible replicons of pTC-F14 and pTFFC2
raised the question of how the replicons of the two sister plasmids had evolved
such that they can now co-exist in the same host cell line. Further incompatibility
testing with the IncQ-like plasmid pIEll08 and the IncQ prototype plasmid
RSF10101R11621R300B determined that pTC-F14 was compatible with pIEI108, but
incompatible with the IncQ prototype plasmid. It was found that the RepB and RepA
replication proteins ofpTF-FC2 and pIEll08 were able to complement the pTC-FI4
orthologs only ifpTC-F14 RepC was present in trans. The RepC protein ofpTC-F14
was thus plasmid-template specific, while the RepA and RepB proteins were less
plasmid-template specific. A five nucleotide possible iteron-discriminating region in
the direct repeats of IncQ-like plasmid oriV regions has been identified (Tietze, E.
(1998) Plasmid 39: 165-181). The iteron sequence ofpTC-F14 differs from pTF-FC2
and pIE 1108 by three nucleotides in this iteron-discriminating region. It was
therefore proposed that co-evolution of the iterons and the RepC protein to a point
where the RepC protein no longer recognizes the iteron sequence of a closely related
sister plasmid is the mechanism by which replicons evolve to become compatible in the same host cell. The incompatibility determinant of the IncQ prototype plasmid
RSFlOlOIR11621R300B was also sought, and subsequently localized to the region
encoding the IncQ prototype plasmid's repAC genes. Interference with the initiation
of pTC-F14 replication by the IncQ prototype plasmid was demonstrated by growth
inhibition of a replication-deficient M13 bacteriophage into which oriVpTC-F14 had
been cloned. Secondly, the IncQ prototype derivative pKE462 displaced a ColEloriVpTC-
F14 construct in complementation assays, and a construct containing only the
pTC-F14 repBAC genes similarly displaced the pKE462 plasmid. As the oriVRSFIOIO
region was not incompatible with a pTC-F14 replicon, this suggested that it was not
the oriV region which was expressing incompatibility, but the products of the IncQ
prototype plasmid repAC genes. It is proposed that incompatibility between pTC-F14
and the IncQ prototype plasmid was the consequence of the repAC gene products
binding to the iterons of the related rep licon, and that these products are unable to
initiate replication. The compatible phenotypes expressed by members of the IncQ
plasmid family indicates the inadequacy of using plasmid incompatibility as a
classification system. Alignment of the amino acid sequences of the three replication
protein orthologs clearly showed that the IncQ plasmid family was divided into two
groups. To account for replication protein relatedness and the incompatibility
phenotype expressed, it is now proposed that that members of the IncQ family be
classified into subdivisions that reflect the different IncQ-like replicons identified in
this study. Investigation of pTC-F14 replicon regulation identified a putative
promoter sequence which is believed to regulate the initiation of a 5.l-5.7-kb
polycistronic transcript that encodes all the replication proteins of the pTC-F14
replicon and the MobB and MobA proteins of the IncP-type mobilization module.
The large polycistronic transcript appears to regulated by the RepB protein of the
pTC-F14 replicon, and is not subject to cross-regulation by related IncQ plasmids.
This suggested that the RepB primase function was not plasmid specific, but that its
regulatory function was replicon specific. A second putative promoter sequence
identified upstream of the pTC-F14 pasAB operon was, however, cross-regulated by
the closely related pTF-FC2 plasmid. The pTC-F14 pas operon encodes two proteins
with high amino acid sequence identity (PasA, 81 %; PasB, 72 %) to the plasmid
addiction system ofpTF-FC2. This is the second time a plasmid addiction system of
this type has been found on an IncQ-like plasmid. / AFRIKAANSE OPSOMMING: Die matig termofiliese (45 to 50°C), hoogs asidofiliese (pH 1.5 to 2.5), chemolitooutotrofiese
Acidithiobaci/lus caldus ras "f' is geïsoleer vanaf 'n biooksiderende
proses wat gebruik word om gekonsentreerde nikkel-erts te behandel. Trans-
Afwisselende Veld Elektroforese (TAVE) analise van totale DNA vanaf die At.
caldus selle, het twee plasmiede van ongeveer 14 en 45-kb. onthul. Die 14-kb
plasmied, genaamd pTC-F14, is gekloneer en deur vervanging van die
kloneringsvektor met 'n kanamisien weerstandsgeen is daar gewys dat hierdie
plasmied in staat is tot outonome replikasie in Escherichia coli. Outonome replikasie
is ook gedemonstreer in Pseudomonas putida en Agrobacterium tumefaciens LBA
4404 wat suggereer dat pTC-F14 'n wye gasheer-reeks plasmied is. Volgorde analise
van die pTC-F14 replikon area het vyf oop leesrame onthul, en 'n replikon organisasie
soortgelyk aan dié van die wye gasheer-reeks IncQ plasmiede. Drie van die oop
leesrame kodeer vir replikasie proteïene met aminosuur volgordes ooreenstemmend
met dié van die IncQ-tipe plasmied pTF-FC2 (RepA, 81%; RepB, 78%; RepC, 74%).
Hierdie hoë vlak van verwantskap stel voor dat die twee replikons vanaf 'n
gemeenskaplike voorouer ontwikkel het. Aangesien naby-verwante replikons
gewoonlik onverenigbaar is, het die verenigbaarheid van die replikons van pTC-F14
en pTF-FC2 die vraag laat onstaan van hoe die replikons van twee susterplasmiede
ontwikkel het, sodat hulle nou gelyktydig in dieselfde gasheer sellyn kan
voortbestaan. Verdere onverenigbaarheid toetsing van die IncQ-tipe plasmied
pIE1108 en die IncQ prototipe plasmied RSF10101R11621R300B, het bepaal dat pTCF14
verenigbaar is met pIE1108, maar onverenigbaar met die IncQ prototipe
plasmied. Daar is gevind dat die RepB en RepA replikasie proteïene van pTF-FC2 en
pIE1108 in staat was om die pTC-F14 ortoloë te komplementeer, slegs as pTC-F14
RepC in trans teenwoordig was. Die RepC proteïen van pTC-F14 is dus plasmiedtemplaat
spesifiek, terwyl die RepA en RepB proteïene minder plasmied-templaat
spesifiek is. 'n Moontlike iteron-onderskeidende vyf-nukleotied area in die direkte
herhalings van die IncQ-tipe plasmied oril/ areas, is geïdentifiseer (Tietze, E. (1998)
Plasmid 39: 165-181). Die iteron volgorde van pTC-F14 verskil van pTF-FC2 en
pIEll08 met drie nukleotiedes in hierdie iteron-onderskeidende area. Om hierdie
rede is daar voorgestel dat ko-evolusie van iterons en die RepC proteïen, tot by 'n punt waar die RepC proteïen nie meer die iteron volgorde van 'n naby-verwante
susterplasmied herken nie, die meganisme is waardeur replikons ontwikkel om
verenigbaar te word in dieselfde gasheersel. Die onverenigbaarheidsbepaler van die
IneQ prototipe plasmied RSFIOIOIR11621R300B is ook ondersoek en gelokaliseer tot
die area wat kodeer vir die IneQ prototipe plasmied se repAC gene. Inmenging met
die inisiasie van pTC-F14 replikasie deur die IneQ prototipe plasmied is
gedemonstreer deur groei vertraging van 'n replikasie-gebrekkige M13 bakteriofaag
waarin die oriVpTC-F14 gekloneer is. Tweedens is die ColEl-oriVpTc-FI4 konstruk
vervang deur die IneQ prototipe-afgeleide pKE462 in komplementasie proewe, en is
die pKE462 plasmied op soortgelyke wyse vervang deur 'n konstruk wat slegs die
pTC-F14 repBAC gene bevat. Aangesien die oriVRSF1010 area nie verenigbaar was met
'n pTC-F14 replikon nie, stel dit voor dat dit nie die oriV area is wat
onverenigbaarheid uitdruk nie, maar die produkte van die IneQ prototipe plasmied se
repAC gene. Dit is voorgestel dat onverenigbaarheid tussen pTC-F14 en die IneQ
prototipe plasmied die gevolg is van die repAC geenprodukte wat bind aan die iterons
van die verwante replikon en dat hierdie produkte nie in staat is om replikasie te
inisieer nie. Die verenigbare fenotipes wat deur die lede van die IneQ plasmied
familie uitgedruk word, dui aan op die ontoereikendheid van die gebruik van plasmied
onverenigbaarheid as 'n klassifikasie sisteem. Vergelyking van die aminosuur
volgordes van die drie replikasie proteïen ortoloë wys duidelik daarop dat die IneQ
plasmied familie in twee groepe verdeel is. Om verantwoording te doen vir die
replikasie proteïen verwantskap en die onverenigbare fenotipe wat uitgedruk is, word
daar nou voorgestel dat die lede van die IneQ familie geklassifiseer word in subafdelings
wat die verskillende IneQ-tipe replikons geïdentifiseer in hierdie studie,
reflekteer. Ondersoek na die pTC-F14 replikon regulering het 'n moontlike promotor
volgorde geïdentifiseer. Daar word gemeen dat hierdie promotor die inisiasie van 'n
5.l-5.7-kb polisistroniese transkrip reguleer, wat kodeer vir al die replikasie proteïene
van die pTC-F14 replikon en die MobB en Mob A proteïene van die IneP-tipe
mobilisasie module. Die groot polisistroniese transkrip blyk om gereguleer te word
deur die RepB proteïen van die pTC-F14 replikon, en word nie gekruis-reguleer deur
die IneQ plasmiede nie. Dit stel voor dat die RepB primase se funksie nie plasmiedspesifiek
is nie, maar dat die reguleerbare funksie replikon-spesifiek is. 'n Tweede
moontlike promotor volgorde wat stroom-op van die pTC-F14 pasAB operon
geïdentifiseer is, is egter gekruis-reguleer deur die pTF-FC2 plasmied. Die pTC-F14 pas operon kodeer vir twee proteïene met hoë aminosuur volgorde verwantskappe
(PasA, 81 %; PasB, 72 %) aan die plasmied-verslaafde sisteem van pTF-FC2. Dit is
die tweede keer dat hierdie tipe plasmied-verslaafde sisteem in 'n IncQ-tipe plasmied
gevind is.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53273 |
| Date | 12 1900 |
| Creators | Gardner, Murray Newell |
| Contributors | Rawlings, D. E., 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 | xiii, 190 p. : ill. |
| Rights | Stellenbosch University |
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