The pXO1 and pXO2 plasmids of B. anthracis are both necessary for full virulence, and understanding the mechanisms by which these plasmids replicate would be helpful in combating anthrax and the spread or use of these plasmids in other systems. A 5-kilobase region of the pXO1 plasmid was cloned into an E. coli vector and replicates when introduced into B. anthracis. Deletion analysis indicated that a 158bp region containing a stem-loop structure contains the origin of replication. Mutational analysis showed that open reading frame 45 (repX) of pXO1 is required for the replication of the miniplasmid in B. anthracis. Interestingly, repX showed limited homology to bacterial FtsZ proteins that are involved in cell division. A mutation in the predicted GTP binding domain of RepX abolished its replication activity. RepX was purified as an MBP- as well as His- N-terminal fusion by overexpression in E. coli and had strong GTPase activity. RepX also bound to DNA weakly and non-specifically.
A potential origin of replication (ori) and replication initiator gene, repS of plasmid pXO2 was cloned into an E. coli vector (pBSCm) which was shown to replicate in B. anthracis, B. cereus, and B. subtilis. The mini pXO2 replicon could not be established in B. subtilis polA mutant, suggesting that DNA Pol I is required for plasmid replication. A countertranscript encoded by the repS promoter region was identified which may control pXO2 copy number by inhibiting repS expression. RepS of pXO2 was overexpressed and purified from E. coli as an MBP fusion at the amino terminal end. DNA binding experiments using double-stranded (ds) and single-stranded (ss) substrates showed that MBP-RepS specifically binds to a 60-bp ds sequence containing the putative pXO2 origin of replication and that a central 20-bp region containing the putative start site for replication and the 5 side of the origin is important for this binding. MBP-RepS also bound to ss DNA non-specifically.
A cell-free system from plasmid-negative B. anthracis cells that promotes robust replication of rolling-circle replicating (RCR) plasmids was developed and adapted to study the replication of plasmid pXO2 in vitro. This system showed that pXO2 replication requires RepS supplied in trans and directional transcription into the origin.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-12182006-111539 |
| Date | 20 December 2006 |
| Creators | Tinsley, Eowyn May |
| Contributors | Roger Hendrix, Martin Schmidt, Saleem Khan, James Carroll, Bruce McClane |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-12182006-111539/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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