Link to the University of Pittsburgh Homepage
Link to the University Library System Homepage Link to the Contact Us Form

Identification and Characterization of the Replicons of the Bacillus anthracis Virulence plasmids pXO1 and pXO2

Tinsley, Eowyn May (2006) Identification and Characterization of the Replicons of the Bacillus anthracis Virulence plasmids pXO1 and pXO2. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

Primary Text

Download (4MB) | Preview


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.


Social Networking:
Share |


Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Tinsley, Eowyn Mayemd41@pitt.eduEMD41
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairKhan, Saleemkhan@mgb.pitt.eduKHAN
Committee MemberMcClane, Brucebamcc@pitt.eduBAMCC
Committee MemberCarroll,
Committee MemberSchmidt, Martinmcs2@pitt.eduMCS2
Committee MemberHendrix, Rogerrhx@pitt.eduRHX
Date: 20 December 2006
Date Type: Completion
Defense Date: 14 December 2006
Approval Date: 20 December 2006
Submission Date: 18 December 2006
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Molecular Virology and Microbiology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Bacillus anthracis; FtsZ; pAMbeta1; pXO1; pXO2; replication; virulence plasmids
Other ID:, etd-12182006-111539
Date Deposited: 10 Nov 2011 20:11
Last Modified: 15 Nov 2016 13:54


Monthly Views for the past 3 years

Plum Analytics

Actions (login required)

View Item View Item