Plasmid pXO1 encodes the tripartite anthrax toxin, which is the major virulence factor of Bacillus anthracis. In spite of the important role of pXO1 in anthrax pathogenesis, very little is known about its replication and maintenance in B. anthracis. We cloned a 5-kb region of the pXO1 plasmid into an Escherichia coli vector and showed that this plasmid can replicate when introduced into B. anthracis. Mutational analysis showed that open reading frame 45 (repX) of pXO1 was 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. Genes almost identical to repX are contained on several megaplasmids in members of the Bacillus cereus group, including a B. cereus strain that causes an anthrax-like disease. Our results identify a novel group of FtsZ-related initiator proteins that are required for the replication of virulence plasmids in B. anthracis and possibly in related organisms. Such replication proteins may provide novel drug targets for the elimination of plasmids encoding the anthrax toxin and other virulence factors.Bacillus anthracis is a gram-positive, spore-forming bacterium that is the etiological agent of anthrax in humans (reviewed in references 12, 19, and 25). B. anthracis is genetically very closely related to Bacillus cereus and Bacillus thuringiensis (13,16,34), and most of the chromosomal virulence-promoting genes of B. anthracis are also present in the latter organisms. A major difference between B. anthracis and related organisms is the presence of two large plasmids, pXO1 and pXO2, which are required for the virulence of this organism (14,19,25,31,36). Recently, a strain of B. cereus has been described that contains a plasmid almost identical to pXO1 and causes an anthrax-like disease (15). The pXO1 plasmid (181.6 kb) encodes the anthrax toxin proteins termed the protective antigen, lethal factor, and the edema factor (10,18,19,25). This plasmid also contains genes that are involved in germination of the spores and genes such as atxA and pagR that regulate the expression of the anthrax toxin and other virulence factors (3,10,19,28). Plasmid pXO1 also contains a number of genes resembling those involved in the horizontal transfer of plasmids (9,19,28), suggesting that this plasmid may mediate its own transfer to other related organisms.Very little is known about the replication properties of the pXO1 plasmid. Studies of the identification of the pXO1 replicon have been hampered since this plasmid does not encode proteins that share significant similarity with known replication initiator proteins encoded by other plasmids (5, 6, 17) (http://www.essex .ac.uk/bs/staff/osborn/DPR_home.htm). Understanding the replication properties of pXO1 is critical for analyzing the potential of this plasmid to replicate and transfer the anthrax toxin-encoding genes in nature.In this study, we describe the isola...

SummaryRepX protein encoded by the pXO1 plasmid of Bacillus anthracis is required for plasmid replication. RepX harbours the tubulin signature motif and contains limited amino acid sequence homology to the bacterial cell division protein FtsZ. Although replication proteins are not known to polymerize, here we show by electron microscopy that RepX undergoes GTPdependent polymerization into long filaments. RepX filaments assembled in the presence of GTPgS were more stable than those assembled in the presence of GTP, suggesting a role for GTP hydrolysis in the depolymerization of the filaments. Light scattering studies showed that RepX underwent rapid polymerization, and substitution of GTP with GTPgS stabilized the filaments. RepX exhibited GTPase activity and a mutation in the tubulin signature motif severely impaired its GTPase activity and its polymerization in vitro. Unlike FtsZ homologues, RepX harbours a highly basic carboxyl-terminal region and exhibits GTP-dependent, non-specific DNA binding activity. We speculate that RepX may be involved in both the replication and segregation of the pXO1 plasmid.

Bacillus anthracis is a gram-positive bacterium that is the etiological agent of anthrax (reviewed in references 18, 24, and 31). There is a high degree of similarity between B. anthracis and members of the Bacillus cereus group (B. cereus, Bacillus thuringiensis, and Bacillus mycoides), with the major differences between these organisms being the presence or absence of two large virulence plasmids, pXO1 and pXO2 (18,19,23,24,31,33,36,39,40). Plasmid pXO1 (181.6 kb) encodes the anthrax toxin proteins termed protective antigen, lethal factor, and edema factor (16,20,23,24,32,33). Plasmid pXO2 (96.2 kb) contains the capA, capB, and capC genes required for capsule biosynthesis and the dep gene involved in the depolymerization of the capsule (14,24,28,32,34,41). In addition, both plasmids carry regulatory genes that control expression of the toxin and capsule genes: atxA and pagR on pXO1 (3,10,17,20,25,30,41,42) and acpA and acpB on pXO2 (11,43).Although pXO1 and pXO2 play central roles in the pathogenesis of anthrax (24,31,44), little is known about the mechanism(s) of replication and copy number control of these plasmids. In culture, the pXO1 plasmid is extremely stable and is rarely cured spontaneously, while the pXO2 plasmid is not as stable and much more likely to be cured (14,24,31). A recent report suggested that differences in pXO2 copy number in naturally occurring strains may, at least in part, be related to differences in virulence (9). pXO1 and pXO2 replication and maintenance are not limited to B. anthracis. Although selftransmission of the plasmids has not been demonstrated, pXO1 and pXO2 can be mobilized into the closely related species B. cereus and B. thuringiensis by conjugative plasmids found in the B. cereus group (2,15,23,24). Interspecies transduction of pXO2 into B. cereus has also been reported (14).The pXO2 plasmid contains sequences that share homology with the replication regions of plasmids of the pAM␤1 family, such as pAW63, pAM␤1, pIP501, and pSM19035, which are found in gram-positive organisms, suggesting that pXO2 also belongs to this plasmid family (4,7,26,34,45). These conjugative plasmids are promiscuous and have a broad host range (7). They replicate by a theta-type mechanism, and their replication proceeds unidirectionally from the origin (6, 7). Sequence alignments have shown that the predicted replication initiator protein of pXO2 termed RepS (ORF-38; 512 amino acids; nucleotides [nt] 34115 to 32580 of pXO2, GenBank accession no. NC_002146) has 96% identity with the Rep63A protein of the B. thuringiensis plasmid pAW63 (34, 45). The RepS protein of pXO2 also has approximately 40% identity with the Rep proteins of plasmids pAM␤1 and pRE25 of Enterococcus faecalis, pIP501 and pSM19035 of Streptococcus agalactiae, and pPLI100 of Listeria innocua on the basis of BLAST alignments (1). Similarly, the putative origin of replication (ori) of pXO2 (nt 32583 to 32524) is highly homologous to the postulated ori of pAW63 (34,45), and the ori of pAM␤1 (4-7, 26, 27).The replication regions o...

PcrA is an essential helicase in gram-positive bacteria, and a gene encoding this helicase has been identified in all such organisms whose genomes have been sequenced so far. The precise role of PcrA that makes it essential for cell growth is not known; however, PcrA does not appear to be necessary for chromosome replication. The pcrA gene was identified in the genome of Bacillus anthracis on the basis of its sequence homology to the corresponding genes of Bacillus subtilis and Staphylococcus aureus, with which it shares 76 and 72% similarity, respectively. The pcrA gene of B. anthracis was isolated by PCR amplification and cloning into Escherichia coli. The PcrA protein was overexpressed with a His 6 fusion at its amino-terminal end. The purified His-PcrA protein showed ATPase activity that was stimulated in the presence of single-stranded (ss) DNA (ssDNA). Interestingly, PcrA showed robust 335 as well as 533 helicase activities, with substrates containing a duplex region and a 3 or 5 ss poly(dT) tail. PcrA also efficiently unwound oligonucleotides containing a duplex region and a 5 or 3 ss tail with the potential to form a secondary structure. DNA binding experiments showed that PcrA bound much more efficiently to oligonucleotides containing a duplex region and a 5 or 3 ss tail with a potential to form a secondary structure than to those with ssDNAs or duplex DNAs with ss poly(dT) tails. Our results suggest that specialized DNA structures and/or sequences represent natural substrates of PcrA in biochemical processes that are essential for the growth and survival of gram-positive organisms, including B. anthracis.Bacillus anthracis is a gram-positive, spore-forming bacteria that is the etiological agent of anthrax in humans (reviewed in references 13, 20, and 27). B. anthracis is a potential biological weapon, and an in-depth understanding of the cellular processes that are important for its growth and survival is critical to combat bioterror agents created on the basis of this and related organisms. DNA helicases are required for critical cellular processes such as DNA replication, transcription, recombination, and repair (3,9,12,(22)(23)(24). Most bacterial species contain several DNA helicases. The DnaB helicase of gramnegative bacteria is necessary for cell survival and is known to be involved in the theta-type replication of the chromosome as well as of several plasmids (6,9,26,33). Gram-positive organisms such as Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, and B. anthracis contain a homolog of the replicative DnaB helicase of Escherichia coli termed DnaC (www.tigr .org). It has been shown that DnaC is required for chromosome replication in B. subtilis and S. aureus (5, 28, 29), and it is highly likely that this is also the case for other gram-positive organisms, including B. anthracis.In addition to DnaC, gram-positive bacteria also contain another helicase, PcrA, which is essential for cell survival in S. aureus and B. subtilis (14,28). PcrA belongs to superfamily I of DNA helicases that s...

Capsule-encoding virulence plasmid pXO2 of Bacillus anthracis is predicted to replicate by a unidirectional theta-type mechanism. To gain a better understanding of the mechanism of replication of pXO2 and other plasmids in B. anthracis and related organisms, we have developed a cell-free system based on B. anthracis that can faithfully replicate plasmid DNA in vitro. The newly developed system was shown to support the in vitro replication of plasmid pT181, which replicates by the rolling-circle mechanism. We also demonstrate that this system supports the replication of plasmid pXO2 of B. anthracis. Replication of pXO2 required directional transcription through the plasmid origin of replication, and increased transcription through the origin resulted in an increase in plasmid replication.In vitro replication systems provide important tools for the study of DNA replication. Rolling-circle (RC)-replicating plasmids are ubiquitous in gram-positive bacteria, including the members of the Bacillus cereus group. B. cereus, Bacillus thuringiensis, and Bacillus mycoides contain indigenous RC-replicating plasmids (2, 12, 18), while RC-replicating plasmids of Staphylococcus aureus such as pT181, pC194, and pE194 can also replicate and be established in Bacillus anthracis (1, 24). Members of this group of organisms also contain large plasmids that presumably replicate by the theta-type mechanism (3, 15, 17, 31, 33-35, 38, 41, 43, 44). B. anthracis contains two large virulence plasmids, pXO1 and pXO2, and related plasmids have also been identified in other members of the B. cereus group (3, 15, 17, 31, 33-35, 38, 41, 43, 44). Plasmid pXO1 (181.6 kb) encodes the anthrax toxin proteins termed the protective antigen, lethal factor, and the edema factor (14,16,24,25,32). Plasmid pXO2 (96.2 kb) contains genes involved in capsule production (24,32).Plasmid pXO2 contains sequences that resemble those present in the replication regions of gram-positive plasmids such as pAM␤1, pAW63, pIP501, and pSM19035, suggesting that pXO2 also belongs to the pAM␤1 family of plasmids (35). These conjugative plasmids replicate by a theta-type mechanism, and their replication proceeds unidirectionally from the origin (9). We have isolated a pXO2 minireplicon containing the repS gene and the origin of replication (ori) (42). The RepS protein of pXO2 is 96% identical to the Rep63A protein of plasmid pAW63 and approximately 40% identical to the Rep proteins of plasmids pAM␤1 and pRE25 of Enterococcus faecalis, pIP501 and pSM19035 of Streptococcus agalactiae, and pPLI100 of Listeria innocua. Similarly, the putative ori of pXO2 (nucleotide [nt] positions 32524 to 32583) is 95% homologous to the postulated ori of pAW63 (34, 44) and has a more limited homology with the ori of pAM␤1 (42). The RepE protein of pAM␤1 has been isolated and shown to bind specifically to double-stranded DNA at the origin and nonspecifically to single-stranded DNA (30). The pAM␤1 ori and the putative ori of pAW63 are located immediately downstream of the RepE coding sequence (8,30,...