Analysis of the mobilization region of the broad host-range IncQ-like plasmid, pTC-F14, and its ability to interact with a related plasmid, pTF-FC2

Van Zyl, Leonardo Joaquim (2003-12)

Thesis (MSc)--University of Stellenbosch, 2003.


ENGLISH ABSTRACT: The 14.2 kb plasmid pTC-FI4 was isolated from the moderately thermophilic (45°- 50°C), highly acidophilic (pH 1.5 to 2.5), chemolithotrophic bacterium Acidithiobacil/us caldus and has a replicon that is closely related to the promiscuous, broad host-range, IncQ-family of plasmids. The region containing the mobilization genes was sequenced and encoded five Mob proteins and an origin of transfer, which are related to the DNA processing (Tral) region of IncPI plasmids, rather than to the three Mob protein systems of the IncQ-l-group plasmids (e.g. plasmids RSFIOIO or R1162). Plasmid pTC-F14 is the third example of an IncQ family plasmid that has five mob genes, with the others being pTF-FC2 and pRAS3.1. The minimal region that was essential for mobilization included the mobA, mobB and the mobC genes as well as the oriT. The mobD and mobE genes were non-essential, but together enhanced the mobilization frequency by approximately 300-fold. The repB gene increased the mobilization frequency but was not essential for mobilization. Mobilization of pTC-F14 between Escherichia coli strains by a chromosomally integrated RP4 plasmid was more than 3500-fold less efficient than the mobilization ofpTF-FC2. When both plasmids were co-resident in the same E. coli host, pTC-FI4 was mobilized at almost the same frequency as pTF-FC2. This enhanced pTC-FI4 mobilization frequency was due to the presence of a combination of the pTF-FC2 mobD and mobE gene products, the functions of which are still unknown. pTF-FC2 could mobilize the oriT of pTC-FI4 whereas pTC-F14 could only mobilize the pTFFC2 oriT if provided with some of the mobilization genes from the pTC-FC2 mobilization region. Unexpectedly either the mobEDC genes or the mobAB genes would allow the mobilization of the pTF-FC2 oriT by pTC-F14 even though there was no common gene between the two subsets. No evidence for any negative effect on the transfer of one plasmid by the related, potentially competitive plasmid was obtained.

AFRIKAANSE OPSOMMING: Die 14.2 kb plasmied, pTC-F14, is uit die matig termofiliese (45°C tot 50°C), hoogs asidofiliese (pH 1.5 tot 2.5), chemolitooutotrofiese bakterium Acidithiobaci/lus caldus geisoleer en beskik oor 'n replikon wat verwant is aan die vanaf die IncQ-familie van plasmiede. Hierdie plasmiede is alom bekend vir hulle promiskuïteit tydens konjugasie asook hul vermoë om in 'n groot aantal verskillende gasheer organismes te kan repliseer. DNA volgorde analise van die mobiliserings area het 'n oordrags oorsprong asook vyf oop leesrame onthul wat nader verwant is aan die DNA prosseserings gene van die Tral area op die IncP 1 plasmiede, as die van die mobiliserings stelsel van die IncQ-l-groep plasmiede. Plasmied pTC-Fl4 is die derde voorbeeld, saam met pTF-FC2 en pRAS3.1, van 'n IncQ-tipe plasmied met 'n vyfgeen mobiliserings sisteem. Die kleinste area op die plasmied nodig vir mobilisering van pTC-Fl4 is bepaal, en het die mobA, mobB en mobC gene sowel as die oordrags oorsprong ingesluit. Saam, was die mobD en mobE gene verantwoordelik vir 'n 300- voud toename in die mobilisasie frekwensie van pTC-Fl4 alhowel die gene nie absoluut nodig was vir mobilisering van die plasmied nie. Die repB geen het ook bygedra tot die frekwensie waarteen die volledige plasmied gemobiliseer was, maar hierdie geen was ook nie nodig vir mobilisering van die pTC-F14 plasmied nie. Die frekwensie waarteen pTC-Fl4 tussen Escherichia coli rasse beweeg het tydens konjugasie, terwyl gebruik gemaak is van 'n chromosomaal geintegreerde RP4 plasmied, was ongeveer 3500-voud laer as die van pTF-FC2. Indien beide pTC-Fl4 en pTF-FC2 in dieselfde E. coli gasheer aangetref word, word beide plasmiede teen ongeveer dieselfde frekwensie gemobiliseer. Die verhoogde frekensie vir pTC-Fl4 was as gevolg van die teenwoordigheid van beide die mobD en mobE gene van die pTF-FC2 plasmied, waarvan die funksies nog onbekend is. Plasmied pTF-FC2 kon die oordrags oorsprong van pTC-Fl4 mobiliseer waarteenoor plasmied pTC-FI4 die oordrags oorsprong vanafpTF-FC2 slegs kon mobiliseer indien een van twee dele van die pTF-FC2 mobiliserings gene voorsien word (al was daar geen oorvleuling tussen die twee nie). Alhoewel die plasmiede moontlik kon kompeteer op die vlak van plasmied oordrag is geen negatiewe kompetesie waargeneem tussen dié twee verwante plasmiede nie.

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