A novel FtsZ-like protein is involved in replication of the anthrax toxin-encoding pXO1 plasmid in Bacillus anthracis

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.     

GTP-dependent polymerization of the tubulin-like RepX replication protein encoded by the pXO1 plasmid of Bacillus anthracis

RepX 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 GTP-dependent polymerization into long filaments. RepX filaments assembled in the presence of GTPgammaS 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 GTPgammaS 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.     

Curing of plasmid pXO1 from Bacillus anthracis using plasmid incompatibility

The large plasmid pXO1 encoding the anthrax toxin is important for the virulence of Bacillus anthracis. It is essential to cure pXO1 from B. anthracis to evaluate its role in the pathogenesis of anthrax infection. Because conventional methods for curing plasmids (e.g., curing agents or growth at elevated temperatures) can induce mutations in the host chromosomal DNA, we developed a specific and reliable method to eliminate pXO1 from B. anthracis using plasmid incompatibility. Three putative replication origins of pXO1 were inserted into a temperature-sensitive plasmid to generate three incompatible plasmids. One of the three plasmids successfully eliminated the large plasmid pXO1 from B. anthracis vaccine strain A16R and wild type strain A16. These findings provided additional information about the replication/partitioning of pXO1 and demonstrated that introducing a small incompatible plasmid can generate plasmid-cured strains of B. anthracis without inducing spontaneous mutations in the host chromosome.     

Transduction and conjugation transfer of the pXO2 plasmid in Bacillus anthracis

The plasmid pXO2 determining the capsule synthesis has been shown to be transfered into the cells of different strains of Bacillus anthracis (STI-1, Sterne, KM33, KM35) by the transducing bacteriophage CP54ant and by mobilization by pAM beta 1 replicon with the frequencies, consequently, n.10(-8) and n.10(-7). The optimal parameters for the selection of clones having acquired the pXO2 plasmid have been defined. Mobilization for conjugational transfer has been demonstrated for the plasmid pXO1 coding for the production of Bacillus anthracis toxin. The dramatic increase of virulence for white mice has been registered for Bacillus anthracis strains having acquired the pXO2 plasmid replicon.     

Identification of self-transmissible plasmids in four Bacillus thuringiensis subspecies.

The transfer of plasmids by mating from four Bacillus thuringiensis subspecies to Bacillus anthracis and Bacillus cereus recipients was monitored by selecting transcipients which acquired plasmid pBC16 (Tcr). Transcipients also inherited a specific large plasmid from each B. thuringiensis donor at a high frequency along with a random array of smaller plasmids. The large plasmids (ca. 50 to 120 megadaltons), pXO13, pXO14, pXO15, and pXO16, originating from B. thuringiensis subsp. morrisoni, B. thuringiensis subsp. toumanoffi, B. thuringiensis subsp. alesti, and B. thuringiensis subsp. israelensis, respectively, were demonstrated to be responsible for plasmid mobilization. Transcipients containing any of the above plasmids had donor capability, while B. thuringiensis strains cured of each of them were not fertile, indicating that the plasmids confer conjugation functions. Confirmation that pXO13, pXO14, and pXO16 were self-transmissible was obtained by the isolation of fertile B. anthracis and B. cereus transcipients that contained only pBC16 and one of these plasmids. pXO14 was efficient in mobilizing the toxin and capsule plasmids, pXO1 and pXO2, respectively, from B. anthracis transcipients to plasmid-cured B. anthracis or B. cereus recipients. DNA-DNA hybridization experiments suggested that DNA homology exists among pXO13, pXO14, and the B. thuringiensis subsp. thuringiensis conjugative plasmids pXO11 and pXO12. Matings performed between strains which each contained the same conjugative plasmid demonstrated reduced efficiency of pBC16 transfer. However, in many instances when donor and recipient strains contained different conjugative plasmids, the efficiency of pBC16 transfer appeared to be enhanced.     

Curing the Plasmid pXO2 from Bacillus anthracis A16 Using Plasmid Incompatibility

Plasmid incompatibility, which has no effect on other plasmids or chromosomal genes, can be used to cure a target plasmid. In this report, we successfully cured the plasmid pXO2 from Bacillus anthracis A16 with a newly constructed, incompatible plasmid pKSV7-oriIV and obtained a new pXO2-cured strain, designated A16PI2. This is the first time that a plasmid was cured from the B. anthracis wild-type strain A16 utilizing this principle, which could be considered as an efficacious method to cure large plasmids.     

Properties and incompatibility behavior of miniplasmids derived from the bireplicon plasmid pCG86

Summary Many plasmids belonging to the F incompatibility groups contain more than one basic replicon. The chimeric plasmid pCG86 is an example of such a multireplicon plasmid. The two basic replicons of pCG86, RepFIIA/FIC and RepFIB have been cloned and re-ligated, the copy numbers of the clones have been determined, and the incompatibility behavior of plasmids containing the ligated replicons and the individual replicons has been studied. The bireplicon plasmids are not expected to be incompatible as recipients with monoreplicon RepFIB or RepFIIA/RepFIC plasmids, since when one replicon is challenged by an incoming replicon, the other should be able to handle the plasmid's replication. In our studies, we found that challenge with either monoreplicon plasmid resulted in incompatibility. This incompatibility was increased in bireplicon plasmids in which RepFIB was duplicated. We conclude that in the bireplicon plasmids, challenging the replication control of one replicon by an incompatible plasmid can interfere with the replication originating from the second replicon.     

Contribution of determinants, located in Bacillus anthracis chromosomes, in realizing the pathogenic properties of the pathogen

Comparative study of virulence of B. anthracis strains harbouring pXO1 and pXO2 plasmids in mice and guinea pigs showed that among six B. anthracis strains, three were 100-1000 times less virulent for guinea pigs. Genetic construction of B. anthracis strains using transduction and conjugation transfer of resident plasmids permitted us to rule out the effects of modified pXO1 and pXO2 replicons and to prove the existence of nonidentified chromosome locuses responsible for the development of an infectious process in anthrax, along with plasmid determinants of virulence.     

Isolation of a minireplicon of the virulence plasmid pXO2 of Bacillus anthracis and characterization of the plasmid-encoded RepS replication protein

A minireplicon of plasmid pXO2 of Bacillus anthracis was isolated by molecular cloning in Escherichia coli and shown to replicate in B. anthracis, Bacillus cereus, and Bacillus subtilis. The pXO2 replicon included (i) an open reading frame encoding the putative RepS replication initiation protein and (ii) the putative origin of replication. The RepS protein was expressed as a fusion with the maltose binding protein (MBP) at its amino-terminal end and purified by affinity chromatography. Electrophoretic mobility shift assays showed that the purified MBP-RepS protein bound specifically to a 60-bp region corresponding to the putative origin of replication of pXO2 located immediately downstream of the RepS open reading frame. Competition DNA binding experiments showed that the 5' and central regions of the putative origin were important for RepS binding. MBP-RepS also bound nonspecifically to single-stranded DNA with a lower affinity.     

Sequence and Organization of pXO1, the Large Bacillus anthracis Plasmid Harboring the Anthrax Toxin Genes

The Bacillus anthracis Sterne plasmid pXO1 was sequenced by random, "shotgun" cloning. A circular sequence of 181,654 bp was generated. One hundred forty-three open reading frames (ORFs) were predicted using GeneMark and GeneMark.hmm, comprising only 61% (110,817 bp) of the pXO1 DNA sequence. The overall guanine-plus-cytosine content of the plasmid is 32.5%. The most recognizable feature of the plasmid is a "pathogenicity island," defined by a 44.8-kb region that is bordered by inverted IS1627 elements at each end. This region contains the three toxin genes (cya, lef, and pagA), regulatory elements controlling the toxin genes, three germination response genes, and 19 additional ORFs. Nearly 70% of the ORFs on pXO1 do not have significant similarity to sequences available in open databases. Absent from the pXO1 sequence are homologs to genes that are typically required to drive theta replication and to maintain stability of large plasmids in Bacillus spp. Among the ORFs with a high degree of similarity to known sequences are a collection of putative transposases, resolvases, and integrases, suggesting an evolution involving lateral movement of DNA among species. Among the remaining ORFs, there are three sequences that may encode enzymes responsible for the synthesis of a polysaccharide capsule usually associated with serotype-specific virulent streptococci.     

Participation of the lytic replicon in bacteriophage P1 plasmid maintenance.

P1 bacteriophage carries at least two replicons: a plasmid replicon and a viral lytic replicon. Since the isolated plasmid replicon can maintain itself stably at the low copy number characteristic of intact P1 prophage, it has been assumed that this replicon is responsible for driving prophage replication. We provide evidence that when replication from the plasmid replicon is prevented, prophage replication continues, albeit at a reduced rate. The residual plasmid replication is due to incomplete repression of the lytic replicon by the c1 immunity repressor. Incomplete repression was particularly evident in lysogens of the thermoinducible P1 c1.100 prophage, whose replication at 32 degrees C remained almost unaffected when use of the plasmid replicon was prevented. Moreover, the average plasmid copy number of P1 in a P1 c1.100 lysogen was elevated with respect to the copy number of P1 c1+. The capacity of the lytic replicon to act as an auxiliary in plasmid maintenance may contribute to the extraordinary stability of P1 plasmid prophage.     

Bacillus anthracis pXO1 Plasmid Sequence Conservation among Closely Related Bacterial Species

The complete sequencing and annotation of the 181.7-kb Bacillus anthracis virulence plasmid pXO1 predicted 143 genes but could only assign putative functions to 45. Hybridization assays, PCR amplification, and DNA sequencing were used to determine whether pXO1 open reading frame (ORF) sequences were present in other bacilli and more distantly related bacterial genera. Eighteen Bacillus species isolates and four other bacterial species were tested for the presence of 106 pXO1 ORFs. Three ORFs were conserved in most of the bacteria tested. Many of the pXO1 ORFs were detected in closely related Bacillus species, and some were detected only in B. anthracis isolates. Three isolates, Bacillus cereus D-17, B. cereus 43881, and Bacillus thuringiensis 33679, contained sequences that were similar to more than one-half of the pXO1 ORF sequences examined. The majority of the DNA fragments that were amplified by PCR from these organisms had DNA sequences between 80 and 98% similar to that of pXO1. Pulsed-field gel electrophoresis revealed large potential plasmids present in both B. cereus 43881 (341 kb) and B. thuringiensis ATCC 33679 (327 kb) that hybridized with a DNA probe composed of six pXO1 ORFs.     

Complete sequence analysis of novel plasmids from emetic and periodontal Bacillus cereus isolates reveals a common evolutionary history among the B. cereus-group plasmids, including Bacillus anthracis pXO1

The plasmids of the members of the Bacillus cereus sensu lato group of organisms are essential in defining the phenotypic traits associated with pathogenesis and ecology. For example, Bacillus anthracis contains two plasmids, pXO1 and pXO2, encoding toxin production and encapsulation, respectively, that define this species pathogenic potential, whereas the presence of a Bt toxin-encoding plasmid defines Bacillus thuringiensis isolates. In this study the plasmids from B. cereus isolates that produce emetic toxin or are linked to periodontal disease were sequenced and analyzed. Two periodontal isolates examined contained almost identical approximately 272-kb plasmids, named pPER272. The emetic toxin-producing isolate contained one approximately 270-kb plasmid, named pCER270, encoding the cereulide biosynthesis gene cluster. Comparative sequence analyses of these B. cereus plasmids revealed a high degree of sequence similarity to the B. anthracis pXO1 plasmid, especially in a putative replication region. These plasmids form a newly defined group of pXO1-like plasmids. However, these novel plasmids do not contain the pXO1 pathogenicity island, which in each instance is replaced by plasmid specific DNA. Plasmids pCER270 and pPER272 share regions that are not found in any other pXO1-like plasmids. Evolutionary studies suggest that these plasmids are more closely related to each other than to other identified B. cereus plasmids. Screening of a population of B. cereus group isolates revealed that pXO1-like plasmids are more often found in association with clinical isolates. This study demonstrates that the pXO1-like plasmids may define pathogenic B. cereus isolates in the same way that pXO1 and pXO2 define the B. anthracis species.     

Sympatric soil communities of Bacillus cereus sensu lato: population structure and potential plasmid dynamics of pXO1- and pXO2-like elements

Eighty soil-borne Bacillus cereus group isolates were collected from two neighbouring geographical sites in Belgium. Their genetic relationships and population structure were assessed using Multilocus sequence typing analysis of five chromosomal genes, while the contribution of extrachromosomal elements to the population dynamics was gauged by the presence, diversity and transfer capacity of pXO1- and pXO2-like plasmids. Globally, the bacterial population displayed a broad diversity, including an important subpopulation of psychrotolerant isolates related to Bacillus weihenstephanensis . pXO1- and pXO2-like replicons were present in 12% and 21% of the isolates, but no Bacillus anthracis -related toxin genes were found. Furthermore, only one of the isolates containing a pXO2-related plasmid was shown to be able to mobilize small non-self-conjugative plasmids. Interestingly, several B. cereus sensu lato isolates displaying the same sequence type were observed to have different plasmid contents, suggesting the occurrence of horizontal gene exchange. Similarly, a number of pXO2-like replicons with identical sequences were found in distinct bacterial isolates, therefore strongly arguing for lateral transfers among sympatric bacteria.     

Functional analysis of three plasmids from Lactobacillus plantarum

Lactobacillus plantarum WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism. The host range of the pWCFS101 replicon includes Lactobacillus species and Lactococcus lactis, while that of the pWCFS102 replicon also includes Carnobacterium maltaromaticum and Bacillus subtilis. The larger plasmid is predicted to replicate via the theta-type mechanism. The host range of its replicon seems restricted to L. plantarum. Cloning vectors were constructed based on the replicons of all three plasmids. Plasmid pWCFS103 was demonstrated to be a conjugative plasmid, as it could be transferred to L. plantarum NC8. It confers arsenate and arsenite resistance, which can be used as selective markers.     

pUb6060: a broad-host-range, DNA polymerase-I-independent ColE2-like plasmid

A ColE2-like, cryptic plasmid, pUB6060, of 5.8 kb has been found in a clinical isolate of Plesiomonas shigelloides. The complete sequence of pUB6060 has been determined and reveals a number of interesting features about the plasmid. The ColE2-like replication locus is linked to a functional ColE1-like mobilization locus. Replication is, unusually for ColE2 replicons, DNA polymerase-I-independent and may involve two, rather than the usual one, plasmid-encoded functions. Additionally, it carries two ORFs encoding products of unknown function. The pUB6060 replicon maintains a moderate plasmid copy number (10 per chromosome copy) and permits replication in diverse gram-negative bacteria.     

Rapid genotypic detection of Bacillus anthracis and the Bacillus cereus group by multiplex real-time PCR melting curve analysis

Bacillus anthracis has four plasmid possible virulence genotypes: pXO1+/pXO2+, pXO1+/pXO2-, pXO1-/pXO2+ or pXO1-/pXO2-. Due to the lack of a specific chromosomal marker for B. anthracis, differentiation of the pXO1-/pXO2- form of B. anthracis from closely related Bacillus cereus group species is difficult. In this study, we evaluate the ability of sspE, pXO1 and pXO2 primers to discriminate individual B. anthracis and the B. cereus group genotypes using multiplex real-time PCR and melting curve analysis. Optimal conditions for successful multiplex assays have been established. Purified DNAs from 38 bacterial strains including 11 strains of B. anthracis and 18 B. cereus group strains were analyzed. Nine of the B. cereus group near-neighbor strains were shown by multilocus sequence typing to be phylogenetically proximate to the B. anthracis clade. We have demonstrated that the four plasmid genotypes of B. anthracis and B. cereus group near-neighbors were differentially and simultaneously discriminated by this assay.