Bacillus anthracis and Bacillus cereus PcrA helicases can support DNA unwinding and in vitro rolling-circle replication of plasmid pT181 of Staphylococcus aureus

Abstract

Replication of rolling-circle replicating (RCR) plasmids in gram-positive bacteria requires the unwinding of initiator protein-nicked plasmid DNA by the PcrA helicase. In this report, we demonstrate that heterologous PcrA helicases from Bacillus anthracis and Bacillus cereus are capable of unwinding Staphylococcus aureus plasmid pT181 from the initiator-generated nick and promoting in vitro replication of the plasmid. These helicases also physically interact with the RepC initiator protein of pT181. The ability of PcrA helicases to unwind noncognate RCR plasmids may contribute to the broad-host-range replication and dissemination of RCR plasmids in gram-positive bacteria.

FIG. 1.

SDS-PAGE analysis of the purified B. cereus PcrA protein. Lanes: U, lysates from uninduced cells; I, lysates from IPTG-induced cells overexpressing the His-PcrA protein; P, B. cereus PcrA protein purified by nickel affinity column chromatography; M, protein molecular mass standards (in kilodaltons).

FIG. 2.

B. anthracis and B. cereus PcrA helicases can unwind plasmid pT181 DNA from a RepC-generated nick. Plasmid pT181cop608 DNA was incubated with 10 ng of different PcrA helicases from different species in the absence (−) or presence (+) of RepC, and the reaction products were analyzed by agarose gel electrophoresis in the presence of ethidium bromide. The PcrA helicases were from S. aureus (Sa), B. anthracis (Ba), B. cereus (Bc), and the K33AQ250R mutant of S. aureus PcrA (Sa-mut). The nickel affinity column fraction obtained by mock purifying proteins from the wild-type E. coli M15 cells (Mock) was tested. The positions of nicked open-circular DNA (OC), supercoiled plasmid DNA (SC), covalently closed relaxed DNA (Rel), and unwound plasmid DNA (U) are indicated to the left of the gel.

FIG. 3.

B. anthracis and B. cereus PcrA helicases support in vitro replication of the heterologous pT181 plasmid. In vitro replication was performed using the RepC protein (+), and cell extracts (Ext) were made from either wild-type (Wt) or pcrA3 mutant S. aureus cells. PcrA helicases from S. aureus (Sa), B. cereus (Bc), and B. anthracis (Ba) were tested. The positions of replication intermediates (RI), open circular DNA (OC), supercoiled pT181cop608 DNA (SC), and single-stranded DNA (SS) are shown to the left of the gel.

FIG. 4.

Interaction between B. anthracis and B. cereus PcrA helicases and RepC. The physical interaction between PcrA and RepC proteins was analyzed by a pull-down assay as described in the text. The eluted fractions from amylose resin columns (either containing [+] or lacking [−] bound MBP-RepC) were probed with either anti-MBP or anti-His₆ monoclonal antibodies (Ab). His-PcrA helicases from S. aureus (Sa), B. anthracis (Ba), and B. cereus (Bc) were used. Equal amounts of protein extracts containing His-PcrA were deposited in the load control lanes (Sa, Ba, and Bc load).The protein extracts were bound to a Ni-nitrilotriacetic acid column, eluted, and then analyzed by SDS-PAGE and immunoblotting. The same amounts of protein extracts were used in the pull-down assays. In lanes lacking MBP-RepC, 1% BSA was used instead.

FIG. 5.

Agarose gel analysis of lysed B. anthracis and B. cereus cells containing the pSA5000 plasmid. (A) Ethidium bromide staining of B. anthracis transformed with pSA5000 DNA. (B) Southern blot analysis of a B. cereus transformant containing plasmid pSA5000. The positions of supercoiled plasmid (SC) and open-circular plasmid (OC) are indicated to the left of the gel.