This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Okinaka, R. T.
Right arrow Articles by Jackson, P. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Okinaka, R. T.
Right arrow Articles by Jackson, P. J.

 Previous Article  |  Next Article 

Journal of Bacteriology, October 1999, p. 6509-6515, Vol. 181, No. 20
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

R. T. Okinaka,1,* K. Cloud,1,dagger O. Hampton,1 A. R. Hoffmaster,2 K. K. Hill,1 P. Keim,3 T. M. Koehler,2 G. Lamke,1,Dagger S. Kumano,1,§ J. Mahillon,4 D. Manter,1,parallel Y. Martinez,1 D. Ricke,1 # R. Svensson,1 and P. J. Jackson1

Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 875451; Department of Biology, Northern Arizona University, Flagstaff, Arizona 86011-56403; Department of Microbiology and Molecular Genetics, University of Texas---Houston Medical School, Houston, Texas 770302; and Laboratoire de Génétique Microbienne---UCL, Louvain-la-Neuve, Belgium4

Received 12 April 1999/Accepted 30 July 1999


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Anthrax, a disease of herbivores and other mammals, including humans, is caused by Bacillus anthracis, a gram-positive, rod-shaped, nonmotile, spore-forming bacterium. Virulence of most B. anthracis strains is associated with two megaplasmids, and strains lacking either plasmid are either avirulent or significantly attenuated. Plasmid pXO2 (60 MDa) carries genes required for the synthesis of an antiphagocytic poly-D-glutamic acid capsule (24, 37, 48, 60, 63, 64). The 110-MDa plasmid pXO1 (61) is required for synthesis of the anthrax toxin proteins, edema factor, lethal factor, and protective antigen. These proteins act in binary combinations to produce the two anthrax toxins: edema toxin (a protective antigen and edema factor) and lethal toxin (a protective antigen and lethal factor) (reviewed in references 41 and 42).

Despite the important roles of pXO1 and pXO2 in B. anthracis virulence, few plasmid-encoded genes have been identified and characterized. Plasmid pXO2 carries three genes required for capsule synthesis (capB, capC, and capA), a gene associated with capsule degradation (dep), and a trans-acting regulatory gene (acpA) (24, 48, 63, 64). Plasmid pXO1 harbors the structural genes for the anthrax toxin proteins [cya (edema factor), lef (lethal factor), and pagA (protective antigen)], as well as two trans-acting regulatory genes (atxA and pagR), a gene encoding a type I topoisomerase (topA), and a recently characterized operon containing three genes whose functions appear to affect germination (12, 21, 26, 31, 38, 50, 55, 62, 65). In addition to the Tox+ phenotype, a number of other phenotypes have been associated with pXO1. pXO1-cured strains exhibit different nutritional requirements on certain minimal media, display enhanced sporulation, and demonstrate altered phage sensitivity compared to pXO1+ strains (57).

Here we report the sequencing, assembly, and annotation of pXO1. Our analysis indicates that this plasmid has the potential to encode 143 proteins. The virulence genes of pXO1 are organized in a manner similar to pathogenicity islands (PAIs) located on the chromosomes of other bacterial pathogens.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Plasmid DNA isolation. The Sterne strain of B. anthracis is pXO1+ pXO2- (50, 56). Plasmid pXO1, isolated and purified from this strain, was kindly provided by Donald Robertson (Department of Chemistry, Brigham Young University, Provo, Utah). The protocol for purification and analysis of pXO1 has been described (34).

DNA sequencing. The sequencing strategy used to assemble the complete plasmid was based on the whole-genome, shotgun approach that has been successfully applied to the sequencing and assembly of several much larger microbial genomes (for an example, see reference 20) and a number of microbial plasmids (18, 23). A small-insert random-fragment library was generated from pXO1 DNA using the following conventional approach. Plasmid DNA was sonicated and size fractionated. Small (2- to 4-kb) DNA fragments were digested with mung bean nuclease to create blunt ends and then ligated into the EcoRV site of Escherichia coli plasmid pT7-Blue (Novagen). Recombinant plasmid DNA was transformed into E. coli DH10B (Life Technologies, Madison, Wis.) by electroporation. Overnight cultures of individual pT7-Blue clones (2 ml) were purified using a 96-well format DNA purification kit (PERFECTprep-96; 5 Prime-3 Prime, Inc., Boulder, Colo.).

Sequences were generated using dye-terminator chemistry (ABI Prism FS; Applied Biosystems, Foster City, Calif.) driven by two pT7-Blue-specific primers complementary to sequences flanking the inserts. Sequencing reactions were analyzed on 36- or 48-cm polyacrylamide gels (4% Burst-Pak custom gel; Owl Scientific, Portsmouth, N.H.) or 5% Long Ranger gels (FMC, Rockland, Maine) using ABI 373 or 377 DNA sequencers (Applied Biosystems).

Sequence assembly and closure. Sequences from randomly selected clones were assembled and edited on an Apple Power Macintosh computer using Sequencher (Gene Codes, Ann Arbor, Mich.). An initial assembly generated ~40 contigs containing both sequence and physical gaps. The sequence gaps were closed primarily by direct walking off of appropriate clones to generate eight contigs containing approximately 180 kb of sequence. The final physical gaps were closed by generating reverse primers to the ends of each contig and systematically linking physical gaps by PCR analysis. A previously constructed pXO1 restriction map (54) was also used to establish appropriate primer pairs. The PCR products were purified with QIAquick PCR Purification Kits (Qiagen, Santa Clarita, Calif.) and sequenced using the protocols outlined above.

Locating open reading frames (ORFs). GeneMark (6) and GeneMark.hmm (43) software programs were used to predict the location of gene boundaries in the pXO1 sequence. GeneMark.hmm uses a specially designed postprocessing ribosome-binding site prediction tool that was modified for a Bacillus subtilis matrix. It considers the lack of the ribosomal protein S1 within the bacilli and the requirement to identify ribosome-binding sites with elevated binding affinities (43). The BLAST and FASTA programs were used to conduct similarity searches against GenBank, EMBL, and Swiss-Prot sequence databases. ScanProsite was used to identify patterns within the Prosite database.

Nucleotide sequence accession number. The complete circular plasmid sequence was entered into GenBank under accession no. AF065404.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Overall features. The Sterne strain of B. anthracis harbors a circular pXO1 plasmid that is 181,654 bp in length with an overall guanine-plus-cytosine content of 32.5%. A total of 143 ORFs were identified using GeneMark and GeneMark.hmm (Fig. 1). BLAST surveys showed that the majority (97) of these ORFs appear to encode proteins of unknown function. Eleven ORFs are predicted to encode proteins with similarity to hypothetical proteins from other organisms (Table 1). Putative functions could be assigned to only 35 ORFs that had significant similarity to proteins of other organisms.


View larger version (27K):
[in this window]
[in a new window]
  		FIG. 1.   Predicted pXO1 ORFs and physical map. The positions of ORFs predicted by GeneMark.hmm are illustrated as arrows along the 181,654-bp circular pXO1 plasmid. The precise start and stop for each predicted ORF and the most likely ribosome binding site are listed in features of pXO1 under GenBank accession no. AF065404. The direction of the arrows indicates the direction of transcription in each ORF relative to all the other ORFs. The gene map illustrates the position of the known toxin genes and the IS1627 elements and shows the relationship of the PAI to the remainder of the pXO1 sequence.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 1.   Putative and characterized pXO1 genes and their functionsa

Gene density. The GeneMark.hmm program predicts that only 110,817 bp of the pXO1 DNA (61%) represents coding regions. The average size of a pXO1 ORF is 610 bp, which is significantly shorter than the average gene size (approximately 1,000 bp) found in most microbial genomes. Other, less-stringent programs that simply separate potentially coding from noncoding regions (e.g., PARSE) indicate that the coding regions may account for as much as 75% of the total pXO1 sequence. The variability in potential coding densities and the shortened average gene size may reflect the presence of nonfunctional gene remnants on the plasmid. These analyses also are consistent with other studies showing a general tendency for plasmids to have larger intergenic spaces (22, 23) than their host microbial genome counterparts, whose gene densities range from 85 to 93% (5, 9, 14, 20, 22, 33, 58).

PAI. The most notable organizational feature of the pXO1 sequence is a large, 44.8-kb region (coordinates 117177 to 162013) whose ends are flanked by two inverted and nearly identical copies of the IS1627 element (Sterne L and Sterne R, originally identified by J. Hornung and C. Thorne as GenBank entries for ORFs 96 and 127, respectively) (Table 1). We designate this region as a PAI because it contains all of the known toxin genes (cya, lef, and pagA) and toxin regulatory elements (atxA and pagR) (see ORFs 107, 109, 110, 119, and 122 in Table 1). The region also contains several putative insertion elements (ISs) and genes encoding integrases and transposases (see ORFs 103, 115, 116, and 120 in Table 1). The pXO1 PAI is predicted to contain a total of 31 ORFs. Fifteen ORFs are hypothetical genes that are either sequences from other organisms for which putative functions have not been assigned or "new" ORFs that do not have similarity to genes in databases. Included in this category are putative genes (ORFs 111 and 118) that were originally identified during the cloning and sequencing of the pagA and atxA genes (64, 65). One ORF is predicted to encode a protein with significant similarity to adenine phosphoribosyl transferase of E. coli (ORF 121). Three ORFs have significant similarity to spore germination response genes from other bacilli (ORFs 112 through 114) (Table 1) (26). In addition, a noncoding region of the PAI (coordinates 158.0 to 158.3) has sequence similarity to the Bacillus cereus gene encoding hemolysin II.

ISs. The class of ORFs with the highest overall similarity to known proteins is composed of 15 putative genes that are related to ISs or genes involved in recombination and integration. These include three homologs of the IS231 sequences commonly associated with the Bacillus thuringiensis crystal toxin genes (46). Three iso-IS231 elements were identified within an 8.5-kb region (coordinates 42029 to 50635) and designated IS231R, -S, and -T (Fig. 1 and Table 1, ORFs 35, 36, and 39). While IS231R and IS231S display an apparent integral organization (a 478-amino-acid [aa] putative protein flanked by 20-bp inverted repeat), IS231S is apparently missing its left extremity. Their lengths varied from 1,604 bp for IS231S to 2,066 bp for IS231T. There is remarkable similarity between the IS231 elements from B. anthracis and those isolated from other members of the B. cereus group. The IS231R DNA sequence is 95% identical to that of IS231E from B. thuringiensis serovar finitimus (IS231T shows 97% identity); yet, IS231R is only 63% identical to its coresident element IS231S. Similarly, IS231S is 67% identical to the IS231Y sequence recently isolated from B. cereus (10b).

As described above, a pair of inverted copies of IS-like elements designated IS1627 (GenBank accession no. U30715 and U30713) flank the region of pXO1 that harbors the putative PAI. The IS150-like elements belong to the IS3 family (46). Comparative restriction analysis of this region in the Sterne strain and in the pXO1 plasmids from several other strains, including Weybridge, Weybridge A, and Vollum, indicates that there has been an inversion in this region (57). In Sterne, the two IS1627 elements are separated by 44,836 bp (coordinates 117177 to 162013) and are contained in nearly exactly duplicated sequences of 1,222 and 1,282 bp. These duplications differ by a 68-bp deletion, several single nucleotide additions or deletions, and a small number of nucleotide changes within the sequences adjacent to the ORFs. The two ORFs within these ISs are identical with the exception of a frameshift difference that causes one of the IS-like elements to be 60 aa longer than the other. Neither of these putative ORFs appears to have a strong ribosome binding site and, as a consequence, the GeneMark.hmm program truncates both ORFs to encode polypeptides containing 83 and 143 aa instead of 214 and 274 aa, respectively. A truncated IS1627 putative transposase is also located immediately adjacent to the right end of the inverted region (ORF 129).

Other ORFs with significant similarity to known proteins normally involved in transposition or mobility of DNA include two reverse transcriptase-like proteins from E. coli and Methanobacterium thermoautotrophicum, respectively, that are often associated with group II intron self splicing (ORFs 07 and 23); a B. subtilis gene, yocA, whose product is similar to a transposon-related protein (ORF 81); three potential integrases (ORFs 18, 103, and 132); a Tn4430-like element (44, 45) that contains two ORFs (ORFs 115 and 116) corresponding to a resolvase and to transposase tnpA, respectively; and finally a putative transposase at ORF 120. The high proportion of sequences with predicted functions in insertion and recombination is suggestive of plasmid plasticity and the possibility of horizontal exchange of DNA among strains or species.

Origin of replication. The pXO1 sequence does not contain ORFs with significant homology to any of the rep genes that have previously been associated with origins of replication in large plasmids of gram-positive organisms (3, 4, 7, 8). Robertson et al. (54) attempted to localize the replication origin by cloning pXO1 DNA fragments into an E. coli plasmid and then transforming the plasmid into B. subtilis. Since E. coli plasmids cannot replicate in Bacillus species, any positive transformants would presumably contain the pXO1 replication origin. This strategy produced several clones that mapped to an 11-kb region that lies between coordinates 86249 and 97209 of the pXO1 sequence (Fig. 1). Sequences within these coordinates contain 10 ORFs, but the predicted products do not show sequence similarity to rep proteins associated with theta-replicating plasmids (15, 32).

Other ORFs with similarity to known genes. Immediately adjacent to the right IS1627 element (coordinates 111374 to 114761) of the PAI are a cluster of ORFs predicted to encode proteins with sequence similarity to hyaluronate synthetase (hasA), UDP-glucose-pyrophosphorylase (gtaB), and UDP-glucose-dehydrogenase (ywqF) of Streptococcus (ORFs 93 to 95). These enzymes participate in the biosynthesis of serotype-specific capsular polysaccharides in streptococci (11, 13, 16, 17). There is no evidence that a polysaccharide capsule is produced by B. anthracis, and the significance of these genes on pXO1 is not evident.

The remaining ORFs with sequences suggesting function include putative genes that bear resemblance to the following: an erythrocyte invasion gene from Plasmodium yoelii (ORF 13); a putative cell division gene, ftxZ, from Pyrococcus horikoshii (ORF 45); an S-layer precursor gene from B. anthracis (ORF 54); a gene (kln) encoding a putative 478-aa secretory protein kinase from Chlorobium limicola with a conserved nucleoside triphosphate-binding motif that is similar to that of VirB11 in Agrobacterium tumefaciens and Bordetella pertussis (ORF 59); a regulatory element (rapC) from B. subtilis (ORF 136); the pagR regulatory gene of B. anthracis (ORF 138); and a gene encoding a thermonuclease precursor/micrococcocal nuclease from Staphylococcus aureus (ORF 141). There are 108 additional ORFs on pXO1, encoding hypothetical proteins for which there are either no database matches or no assigned functions.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The most recognizable feature of the B. anthracis Sterne megaplasmid pXO1 is a putative PAI (27) that houses all three toxin genes and all of their known regulatory elements. Two IS1627 elements (ORFs 96 and 127) mark the molecular boundaries of this 44.8-kb region. Thorne (57) provided evidence that this region can behave as a mobile and distinct unit; the 44.8-kb region is inverted in pXO1 plasmids originating from two different strains of B. anthracis. The pXO1 PAI lacks other characteristics common to PAIs of other organisms. The guanine-plus-cytosine content of the pXO1 PAI (30%) does not differ significantly from that of the rest of the plasmid (32.5%) or the host genome (33%) (52). Also, there is no apparent difference in codon usage between the PAI and non-PAI regions (data not shown). These observations suggest that the majority of the pXO1 sequences must have evolved from backgrounds that are similar to this class of bacilli or related organisms.

The pXO1 sequence does not contain a replication initiator-like protein (repA) and typical reiterated sequences (iterons) containing clusters of direct and inverted repeats that define origins of replication in most theta-replicating plasmids (15, 32, 39). An 11-kb region of the plasmid, corresponding to coordinates 86249 to 97209, has been reported to contain an origin of replication (54). None of the ORFs within this region are predicted to encode proteins with functions related to rep or inc functions in other organisms. Theta-replicating plasmids that do not have iterons or encode proteins like the product of repA (15) include the prototype plasmid ColE1 (reviewed in reference 39) and the B. subtilis plasmid pLS20 (49). ColE1 requires the host DNA polymerase I to initiate replication while replication of pLS20 is independent of polymerase I activity. Although pXO1 has sequences that resemble dnaA boxes and replication termination signals, none of these sequences are tightly clustered within any particular region. Searches for palindromes and secondary structures reveal several sites where palindromes exist in small clusters, but a putative origin cannot be assigned primarily on this basis. These results suggest that pXO1 may have a rare and perhaps unique origin of replication.

The high number of IS-like elements on pXO1 suggests that the PAI and perhaps other regions within this plasmid may have evolved by mechanisms involving duplication, inversion, and deletion. A similar phenomenon appears to have occurred in the Rhizobium 536-kb megaplasmid (from strain NGR234) that has 18% of its sequence in the form of mosaic sequences and ISs (23). Families of transposable elements on these large bacterial plasmids may have driven the movement and evolution of genes with functions related to pathogenicity and symbiosis throughout different species.

The presence of 15 ORFs with similarity to transposases, integrases, and recombinases suggests that pXO1 is a plasmid with a high degree of genetic plasticity. However, the genomes of B. anthracis isolates from diverse origins show extremely low levels of genetic variation at the molecular level (36). It has been argued previously that the severe molecular monomorphism of B. anthracis is the result of either a very recent origin of this species or population size constrictions or bottlenecks associated with the spread of this disease. Due to the lack of other genetic variation, structural rearrangements as observed in pXO1 may play a more important evolutionary role than in other organisms. Indeed, the pXO1 genes encoding activities related to recombination could act in trans to increase structural changes in both the B. anthracis chromosome and in pXO2. Rapid evolutionary change could benefit a pathogen, and genetic linkage of trans-acting recombinases to toxin genes could be an evolved situation.

The Lyme disease spirochete, Borrelia burgdorferi, contains at least 17 linear and circular plasmids, whose combined length exceeds 500,000 bp (22). There is one striking similarity between these plasmids and pXO1. The average size of ORFs within the 11 linear Borrelia plasmids is 507 bp. The average size of ORFs in pXO1 is 610 bp. A recent analysis of the Borrelia plasmids indicates that a significant portion of the plasmid genes are damaged due to a myriad of recombinational events between like plasmids (10). The short lengths of the Borrelia plasmid ORFs are apparently due to this decay process, resulting in the accumulation of pseudogenes. It is not clear whether pXO1 is evolving by similar processes. Perhaps a subset of nonfunctional genes within pXO1 are being driven into extinction but are currently trapped in the slow evolution of B. anthracis.

Among the pXO1 ORFs with a high degree of similarity to known genes are two clusters of genes whose potential expression and function could affect the virulence and life cycle of B. anthracis. A cluster of three genes, gerXA, gerXB, and gerXC (ORFs 112, 113, and 114) (26), encode proteins with sequence similarities to proteins involved in the spore germination response in B. subtilis. In B. subtilis, each of four germination response operons, gerA, gerB, gerK, and yfk, encodes three genes (51, 66). For example, the gerA operon contains gerA, gerAB, and gerAC (51). The gerA genes are involved in the germination response to L-alanine. The gerB and gerK loci are involved in the response to a combination of asparagine, glucose, fructose, and KCl. The pXO1-encoded ger genes have recently been shown to belong to a single operon whose function appears to influence the germination rate of B. anthracis spores in a murine alveolar macrophage assay (25, 26). These plasmid-borne gene products appear to facilitate the germination of the bacterial spore at sites within certain mammalian target cells.

Another cluster (ORFs 93, 94, and 95) is a set of three genes that are similar to genes involved in the synthesis of the serotype-specific polysaccharide capsule of virulent group A streptococci. The streptococcal genes hasA, hasB, and hasC are transcribed in an operon and encode hyaluronate synthase, UDP-glucose dehydrogenase, and UDP-glucose pyrophosphorylase, respectively (11, 13, 16, 17). The three corresponding pXO1 genes closely resemble the streptoccal genes (Table 1), except that the order of the pXO1 genes is A, C, B. There are no reports of a polysaccharide capsule in B. anthracis. Genes on the other B. anthracis megaplasmid, pXO2, facilitate synthesis of a D-glutamic acid capsule by virulent strains (60). Therefore, pXO1 ORFs 93, 94, and 95 may represent another example of nonfunctional genes that have yet to decay away.

pXO1 contains three IS231 elements with significant homology to their counterparts in the B. cereus subgroup (46). IS231 is a group of ISs originally identified in large plasmids from the entomopathogen B. thuringiensis. These B. thuringiensis ISs are structurally associated with the insecticidal partial -endotoxin genes and other transposable elements. They harbor two terminal inverted repeats and encode a single transposase related to that found in IS1151 from Clostridium perfringens and the IS4 family of ISs. Following transposition, IS231A generates duplications of between 10 and 12 bp at preferred target sites (46, 53). A recent study of the occurrence and diversity of ISs among B. cereus members shows that, of all the elements tested, IS231 displayed the most widespread distribution and variability (29, 40). Detailed structural and/or functional analyses of several iso-IS231 sequences have since shown that this diversity is not only related to the DNA sequence but also to a modular assemblage of these entities (10b). The presence of IS231 elements in pXO1 confirms previous reports of the existence of these elements in B. anthracis (30). Recent PCR and sequencing data also suggests that IS231-like elements exist on the chromosome of B. anthracis and in the capsule containing plasmid pXO2 (10b). The strong homology (more than 95% identity) between two of the IS231 elements from B. anthracis and IS231E from B. thuringiensis serovar finitimus indicates that either direct exchanges occurred between these hosts or these species at one time shared common genomic pools.

Another observation in pXO1 is the clustering of these three IS231 elements within an 8.5-kb region. This is reminiscent of other IS231 groupings within itself and in association with the class II transposon Tn4430 in B. thuringiensis (44). Mahillon and Chandler (47) recently proposed several mechanisms that could account for this behavior, including the successive insertion of related ISs into a highly preferred region. Insertion specificity appears to have relied on the DNA sequence and structural conformation of the target site for IS231A (28). pXO1 also harbors a Tn4430-like element (ORFs 115 and 116) that spans 3.7 kb and includes a Tn1546-like resolvase and a Tn21-like transposase. However, this element is not linked to the IS231 cluster but is located directly adjacent to the known toxin genes.

Large megaplasmids (50 to 500 kb) that confer selective advantage on their hosts are common throughout microbial communities. Examples include the TOL family of plasmids that allow Pseudomonas species to metabolize toluene and other toxic organic chemicals (2) and the 536-kb plasmid pNGR234a of Rhizobium that promotes the symbiotic relationship between this species and a variety of legumes (23). Many megaplasmids are also known to carry virulence factors. Several gram-positive pathogens, including C. perfringens and B. thuringiensis, harbor such plasmids. Diverse strains of C. perfringens are characterized by their ability to produce numerous extracellular toxins. Genes encoding certain of these toxins are located on the bacterial chromosome, but others are often housed in megaplasmids ranging in size from 90 to 130 kb (19, 35). In B. thuringiensis, at least 50 different insecticidal partial -endotoxins have been identified on plasmids ranging in size from 60 to 300 kb (1). Clearly, the presence of the structural genes encoding the anthrax toxin proteins on pXO1 results in selection for plasmid maintenance by B. anthracis during infection. However, it is not clear if pXO1 confers a selective advantage on B. anthracis in nonhost environments. Spontaneous loss of pXO1 during growth in laboratory medium is rare. Although pXO1- soil isolates of B. anthracis are uncommon, all environmental strains in our collection were isolated from soil collected near diseased animals (36). It is possible that pXO1- isolates exist in other soil samples. Certain pXO1-associated phenotypes have been noted, but they have not been associated with specific genes. These include altered sensitivity to certain bacteriophages, different nutritional requirements for growth on certain minimal media, and altered sporulation characteristics. Study of the numerous pXO1 ORFs that do not have obvious similarity to known genes may lead to a better understanding of these and other phenotypes.


    ACKNOWLEDGMENTS

We acknowledge the technical support of Lakshmi Pillai, Lena Martinez, and James Fulwyler.

This work was supported by the Office of Non-Proliferation and National Security, U.S. Department of Energy. Work by A.R.H. and T.M.K. was supported by Public Health Service Grant AI33537 from the National Institutes of Health.


    FOOTNOTES

* Corresponding author. Mailing address: Life Sciences Division, Los Alamos National Laboratory, M888, Los Alamos, NM 87545. Phone: (505) 667-2743. Fax: (505) 665-3024. E-mail: okinaka{at}telomere.lanl.gov.

dagger Present address: University of Colorado Health Science Center, Denver, CO 80220.

Dagger Present address: Mayo Medical School, Rochester, MN 55905.

§ Present address: Department of Molecular and Cell Genetics, Tottori University, Yonago, Tottori 683, Japan.

parallel Present address: College of Forestry, Oregon State University, Corvallis, OR 97331.

# Present address: Nevarus Agricultural Discovery Institute, San Diego, CA 92121.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

1. Aronson, A. I. 1993. Insecticidal toxins, p. 953-963. In A. L. Sonenshein (ed.), Bacillus subtilis and other gram-positive bacteria. American Society for Microbiology, Washington, D.C..
2. Assinder, S. J., and P. A. Williams. 1990. The TOL plasmids: determinants of the catabolism of toluene and the xylenes. Adv. Microb. Physiol. 31:1-69[Medline].
3. Baum, J., and M. Gilbert. 1991. Characterization and comparative sequence analysis of replication origins from three large Bacillus thuringiensis plasmids. J. Bacteriol. 173:5280-5289[Abstract/Free Full Text].
4. Baum, J., and J. Gonzalez. 1992. Mode of replication, size and distribution of naturally occurring plasmids in Bacillus thuringiensis. FEMS Microbiol. Lett. 96:143-148.
5. Blattner, F., G. Plunkett, C. Bloch, N. Perna, V. Burland, M. Riley, J. Collado-Vides, J. Glasner, C. Rode, G. Mayhew, G. Jason, N. Davis, H. Kerkpatrick, M. Goeden, D. Rose, B. Mau, and Y. Shao. 1997. The complete genome sequence of Escherichia coli K-12. Science 277:1453-1462[Abstract/Free Full Text].
6. Borodovsky, M., and J. McIninch. 1993. GeneMark: parallel gene recognition for both DNA strands. Comput. Chem. 17:123-133.
7. Brantl, S., D. Behnke, and J. Alonso. 1990. Molecular analysis of the replication region of the conjugative Streptococcus agalactiae plasmid pIP501 in Bacillus subtilis. Comparison with plasmids pAMb1 and pSM19035. Nucleic Acids Res. 18:4783-4789[Abstract/Free Full Text].
8. Braund, C., E. Le Chatelier, S. Ehrlich, and L. Janniere. 1993. A fourth class of theta-replicating plasmids: the pAM beta 1 family from gram-positive bacteria. Proc. Natl. Acad. Sci. USA 90:11668-11672[Abstract/Free Full Text].
9. Bult, C., O. White, G. Olsen, L. Zhou, R. D. Fleischmann, G. G. Sutton, J. A. Blake, L. FitzGerald, R. Clayton, J. Gocayne, A. Kerlavage, B. Dougherty, J. Tomb, M. Adams, C. Reich, R. Overbeek, E. Kerkness, K. Weinstock, J. Merrick, A. Glodek, J. Scott, N. Geoghagen, and J. Venter. 1996. Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science 273:1058-1073[Abstract].
10. Casjens, S., W. M. Huang, M. Robertson, N. Palmer, J. Peterson, G. Sutton, and C. Fraser. 1998. Uncontrolled rearrangements among Borrelia linear plasmids? Microbial and comparative genomics, p. C-12. In In Abstracts of the 3rd Conference on Microbial Genomes.
10a. Chen, Y., et al. Unpublished data.
10b. Chen, Y., R. Okinaka, and J. Mahillon. Unpublished data.
11. Crater, D., B. Dougherty, and I. van de Rijn. 1995. Molecular characterization of hasC from an operon required for hyaluronic acid synthesis in group A streptococci. Demonstration of UDP-glucose pyrophophorylase activity. J. Biol. Chem. 270:28676-28680[Abstract/Free Full Text].
12. Dai, Z., J.-C. Sirard, M. Mock, and T. Koehler. 1995. The atxA gene product activates transcription of the anthrax toxin genes and is essential for virulence. Mol. Microbiol. 16:1171-1181[Medline].
13. DeAngelis, P. L., J. Papaconstantinou, and P. H. Weigel. 1993. Molecular cloning, identification, and sequence of the hyaluronan synthase gene from group A Streptococcus pyrogenes. J. Biol. Chem. 268:19181-19184[Abstract/Free Full Text].
14. Deckert, G., P. Warren, T. Gaasterland, W. Young, A. Lenox, D. Graham, R. Overbeek, M. Snead, M. Keller, M. Aujay, R. Huber, R. Feldman, J. Short, G. Olsen, and R. Swanson. 1998. The complete genome of the hyperthermophilic bacterium Aquifex aeolicus. Nature 392:353-358[Medline].
15. del Solar, G., R. Giraldo, M. J. Ruiz-Echevarria, M. Espinosa, and R. Diaz-Orejas. 1998. Replication and control of circular bacterial plasmids. Microbiol. Mol. Biol. Rev. 62:434-464[Abstract/Free Full Text].
16. Dougherty, B., and I. van de Rijn. 1993. Molecular characterization of hasB from an operon required for hyaluronic acid synthesis in group A streptococci. Demonstration of UDP-glucose dehydrogenase activity. J. Biol. Chem. 268:7118-7124[Abstract/Free Full Text].
17. Dougherty, B., and I. van de Rijn. 1994. Molecular characterization of hasA from an operon required for hyaluronic acid synthesis in group A streptococci. J. Biol. Chem. 269:169-175[Abstract/Free Full Text].
18. Dougherty, B., C. Hill, J. Weidman, D. Richardson, J. Venter, and R. Ross. 1998. Sequence and analysis of the 60 kb conjugative bacteriocin-producing plasmid pMRC01 from Lactococcus lactis DPC3147. Mol. Microbiol. 29:1029-1038[Medline].
19. Dupy, B., G. Daube, M. R. Popoff, and S. T. Cole. 1997. Clostridium perfringens urease genes are plasmid borne. Infect. Immun. 65:2313-2320[Abstract].
20. Fleischmann, R., M. Adams, O. White, R. Clayton, E. Kirkness, A. Kerlavage, C. Bult, J.-F. Tomb, B. Dougherty, K. McKenney, G. Sutton, W. FitzHugh, C. Fields, J. Gocayne, J. Scott, R. Shirley, L. Liu, A. Glodek, J. Kelley, J. Weidman, C. Philips, T. Spriggs, E. Hedblom, M. Cotton, T. Utterback, M. Hanna, D. Nguyen, D. Saudek, R. Barndon, L. Fine, J. Fritchman, J. Fuhrmann, N. Geoghagen, C. Gnehm, L. McDonald, K. Small, C. Fraser, H. Smith, and J. Venter. 1995. Whole-genome random sequencing and assembly of Haemophilus influenzae RD. Science 269:496-512[Abstract/Free Full Text].
21. Fouet, A., J.-C. Sirard, and M. Mock. 1994. Bacillus anthracis pXO1 virulence plasmid encodes a type DNA topoisomerase. Mol. Microbiol. 11:471-479[Medline].
22. Fraser, C., S. Casjens, W. M. Huang, G. G. Sutton, R. Clayton, R. Lathigra, O. White, K. A. Ketchum, R. Dodson, E. K. Hickeyt, M. Gwinn, B. Dougherty, J.-F. Tomb, R. K. Fleischmann, D. Richardson, J. Peterson, A. R. Kerlavage, J. Quackenbush, S. Salzberg, M. Hanson, R. van Vugt, N. Palmer, M. D. Adams, J. Gocayne, J. Weidman, T. Utterback, L. Watthey, L. McDonald, P. Artiach, C. Bowman, S. Garland, C. Fujii, M. D. Cotton, K. Horst, K. Roberts, B. Hatch, H. O. Smith, and J. C. Venter. 1997. Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature 390:580-588[Medline].
23. Frelberg, C., R. Fellay, A. Balroch, W. Broughton, A. Rosenthal, and X. Perret. 1997. Molecular basis of symbiosis between Rhizobium and legumes. Nature 387:394-401[Medline].
24. Green, B. D., D. L. Battisti, T. M. Koehler, C. B. Thorne, and B. E. Ivins. 1985. Demonstration of a capsule plasmid in Bacillus anthracis. Infect. Immun. 49:291-297[Abstract/Free Full Text].
25. Guidi-Rontani, C., M. Weber-Levy, E. Labruyere, and M. Mock. 1999. Germination of Bacillus anthracis spores with alveolar macrophages. Mol. Microbiol. 31:9-17[Medline].
26. Guidi-Rontani, C., Y. Pereira, S. Ruffie, J.-C. Sirard, M. Weber-Levy, and M. Mock. 1999. Identification and characterisation of a germination operon on the virulence plasmid pXO1 of Bacillus anthracis. Mol. Microbiol. 33:407-414[Medline].
27. Hacker, J., G. Blum-Oehler, I. Muhldorfer, and H. Tschape. 1997. Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution. Mol. Microbiol. 23:1089-1097[Medline].
28. Hallet, B., R. Rezsöhazy, J. Mahillon, and J. Delcour. 1994. IS231A insertion specificity: consensus sequence and DNA bending at the target site. Mol. Microbiol. 14:131-139[Medline].
29. Helgason, E., D. Caugant, M.-M. Lecadet, Y. Chen, J. Mahillon, A. Lövgren, I. Hegna, K. Kvaløy, and A.-B. Kolstø. 1998. Genetic diversity of Bacillus cereus/B. thuringiensis isolates from natural sources in Norway. Curr. Microbiol. 37:80-87[Medline].
30. Henderson, I., D. Yu, and P. C. Turnbull. 1995. Differentiation of Bacillus anthracis and other "Bacillus cereus group" bacteria using IS231-derived sequences. FEMS Microbiol. Lett. 128:113-118[Medline].
31. Hoffmaster, A. R., and T. M. Koehler. 1999. Autogenous regulation of the Bacillus anthracis pag operon. J. Bacteriol. 181:4485-4492[Abstract/Free Full Text].
32. Janniere, L., A. Gruss, and S. Ehrlich. 1993. Plasmids, p. 625-644. In A. Sonenshein, J. Hoch, and R. Losick (ed.), Bacillus subtilis and other gram-positive bacteria. American Society for Microbiology, Washington, D.C..
33. Kaneko, T., S. Sato, H. Kotani, A. Tanaka, E. Asamizu, Y. Nakamura, N. Miyajima, M. Hirosawa, M. Sugiura, S. Sasamoto, T. Kimura, T. Hosouchi, A. Matsuno, A. Muraki, N. Nakzaki, K. Naruo, S. Okumura, S. Shimpo, C. Takeuchi, T. Wada, A. Watanabe, M. Yamada, M. Yasuda, and S. Tabata. 1996. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res. 3:109-136[Abstract].
34. Kaspar, R., and D. Robertson. 1987. Purification and physical analysis of Bacillus anthracis plasmids pXO1 and pXO2. Biochem. Biophys. Res. Commun. 149:362-368[Medline].
35. Katayama, S., B. Dupuy, G. Daube, B. China, and S. T. Cole. 1996. Genome mapping of Clostridium perfringens strains with I-CeuI shows many virulence genes to be plasmid-borne. Mol. Gen. Genet. 251:720-726[Medline].
36. Keim, P., A. Kalif, J. Schupp, K. Hill, S. E. Travis, K. Richmond, D. M. Adair, M. Hugh-Jones, C. R. Kuske, and P. Jackson. 1997. Molecular evolution and diversity in Bacillus anthracis as detected by amplified fragment length polymorphism markers. J. Bacteriol. 179:818-824[Abstract/Free Full Text].
37. Keppie, J., P. W. Harris-Smith, and H. Smith. 1963. The chemical basis of the virulence of Bacillus anthracis. IX. Its aggressins and their mode of action. Br. J. Exp. Pathol. 44:446-453.
38. Koehler, T. M., Z. Dai, and M. Kaufman-Yarbray. 1994. Regulation of the Bacillus anthracis protective antigen gene: CO2 and a trans-acting element activate transcription from one of the two promoters. J. Bacteriol. 176:586-595[Abstract/Free Full Text].
39. Kornberg, A., and T. A. Baker. 1992. DNA replication, 2nd ed. W. H. Freeman and Company, New York, N.Y.
40. Léonard, C., Y. Chen, and J. Mahillon. 1997. Diversity and differential distribution of IS231, IS232 and IS240 among Bacillus cereus, B. thuringiensis, and B. mycoides. Microbiology 143:2537-2547[Abstract/Free Full Text].
41. Leppla, S. H. 1991. The anthrax toxin complex, p. 277-298. In J. Alouf, and J. Freer (ed.), Sourcebook of bacterial protein toxins. Academic Press, New York, N.Y.
42. Leppla, S. H. 1995. Anthrax toxins, p. 543-572. In J. Moss, B. Iglewski, M. Vaughn, and A. T. Tu (ed.), Bacterial toxins and virulence factors in disease. Marcel Dekker, New York, N.Y.
43. Lukshin, A., and M. Borodovsky. 1998. GeneMark.hmm: new solutions for gene finding. Nucleic Acids Res. 26:1107-1115[Abstract/Free Full Text].
44. Mahillon, J., J. Seurinck, J. Delcour, and M. Zabeau. 1987. Cloning and nucleotide sequence of different iso-IS231 elements and their structural association with the Tn4430 transposon in Bacillus thuringiensis. Gene 51:187-196[Medline].
45. Mahillon, J., and D. Lereclus. 1988. Structural and functional analysis of Tn4430: identification of an integrase-like protein involved in the co-integrate-resolution process. EMBO J. 7:1515-1526[Medline].
46. Mahillon, J., R. Rezsöhazy, B. Hallet, and J. Delcour. 1994. IS231 and other Bacillus thuringiensis elements: a review. Genetica 93:13-26[Medline].
47. Mahillon, J., and M. Chandler. 1998. Insertion sequences. Microbiol. Mol. Biol. Rev. 62:725-774[Abstract/Free Full Text].
48. Makino, S., I. Uchida, N. Terakado, C. Ssakawa, and M. Yoshikawa. 1989. Molecular characterization and protein analysis of the cap region, which is essential for encapsulation in Bacillus anthracis. J. Bacteriol. 171:722-730[Abstract/Free Full Text].
49. Meijer, W. J. J., A. J. de Boer, S. van Tongeren, G. Venema, and S. Bron. 1995. Characterization of the replication region of the Bacillus subtilis plasmid pLS20: a novel type of replicon. Nucleic Acids Res. 23:3214-3224[Abstract/Free Full Text].
50. Mikesell, P., B. E. Ivins, J. D. Ristroph, and T. M. Dreier. 1983. Evidence for plasmid-mediated toxin production in Bacillus anthracis. Infect. Immun. 39:371-379[Abstract/Free Full Text].
51. Moir, A., E. H. Kemp, C. Robinson, and B. M. Corfe. 1994. The genetic analysis of bacterial spore germination. J. Appl. Bacteriol. 77:9S-16S[Medline].
52. Priest, F. 1993. Systematics and ecology of Bacillus, p. 3-33. In A. Sonenshein, J. Hoch, and R. Losick (ed.), Bacillus subtilis and other gram-positive bacteria. American Society for Microbiology, Washington, D.C..
53. Rezsöhazy, R., B. Hallet, J. Delcour, and J. Mahillon. 1993. The IS4 family of insertion sequences: evidence for a conserved transposase motif. Mol. Microbiol. 9:1283-1295[Medline].
54. Robertson, D., T. Bragg, S. Simpson, R. Kaspar, W. Xie, and M. Tippetts. 1990. Mapping and characterization of the Bacillus anthracis plasmids pXO1 and pXO2. Salisbury Med. Bull. 68(Suppl.):55-58.
55. Robertson, D. L., M. T. Tippetts, and S. H. Leppla. 1988. Nucleotide sequence of the Bacillus anthracis edema factor gene (cya): a calmodulin-dependent adenylate cyclase. Gene 73:363-371[Medline].
56. Sterne, M. 1939. The use of anthrax vaccines prepared from avirulent (unencapsulated) variants of Bacillus anthracis. Onderstepoort J. Vet. Sci. Anim. Ind. 13:307-312.
57. Thorne, C. 1993. Bacillus anthracis, p. 113-124. In A. L. Sonenshein (ed.), Bacillus subtilis and other gram-positive bacteria. American Society for Microbiology, Washington, D.C..
58. Tomb, J.-F., O. White, A. Kerlavage, R. Clayton, G. Sutton, R. Fleischmann, K. Ketchum, H. Klenk, S. Gill, B. Dougherty, K. Nelson, J. Quackenbush, L. Zhou, E. Kerkness, S. Peterson, B. Loftus, D. Richardson, R. Dodson, H. Khalak, A. Glodek, K. McKenney, L. Fitzegerald, N. Lee, M. Adams, E. Hickey, D. Berg, J. Gocayne, T. Utterback, J. Peterson, J. Kelley, M. Cotton, J. Weldman, C. Fujii, C. Bowman, L. Watthey, E. Wallin, W. Hayes, M. Borodovsky, P. Karp, H. Smith, C. Fraser, and J. Venter. 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:539-547[Medline].
59. Tomcsik, J. 1956. Bacterial capsules and their relation to the cell wall. In E. T. C. Spooner, and B. A. D. Stocker (ed.), Bacterial anatomy. Cambridge University Press, Cambridge, United Kingdom.
60. Uchida, I., T. Sekizaki, K. Hashimoto, and N. Terakado. 1985. Association of the encapsulation of Bacillus anthracis with a 60 megadalton plasmid. J. Gen. Microbiol. 131:363-367[Abstract/Free Full Text].
61. Uchida, I., K. Hashimoto, and N. Terakado. 1986. Virulence and immunogenicity in experimental animals of Bacillus anthracis strains harbouring or lacking 110 MDa and 60 MDa plasmids. J. Gen. Microbiol. 132:557-559[Abstract/Free Full Text].
62. Uchida, I., J. Hornung, C. Thorne, K. Klimpel, and S. Leppla. 1993. Cloning and characterization of a gene whose product is a trans-activator of anthracis toxin synthesis. J. Bacteriol. 175:5329-5338[Abstract/Free Full Text].
63. Uchida, I., S. Makino, C. Sasakawa, M. Yoshikawa, C. Sugimoto, and N. Terakado. 1993. Identification of a novel gene, dep, associated with depolymerization of the capsular polymer in Bacillus anthracis. Mol. Microbiol. 9:487-496[Medline].
64. Vietri, N., R. Marrero, T. Hoover, and S. Welkos. 1995. Identification and characterization of a trans-activator involved in the regulation of encapsulation by Bacillus anthracis. Gene 152:1-9[Medline].
65. Welkos, S. L., J. R. Lowe, F. Eden-McCutchan, M. Vodkin, S. H. Leppla, and J. J. Schmidt. 1988. Sequence and analysis of the DNA encoding protective antigen of Bacillus anthracis. Gene 69:287-300[Medline].
66. Yamamoto, H., S. Uchiyama, F. A. Nugroho, and J. Sekiguchi. 1997. Cloning and sequencing of a 35.7 kb in the 70°-73° region of the Bacillus subtilis genome reveal genes for a new two-component system, three spore germination proteins, an iron uptake system and a general stress response protein. Gene 194:191-199[Medline].

Journal of Bacteriology, October 1999, p. 6509-6515, Vol. 181, No. 20
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Ebrahimi, C. M., Kern, J. W., Sheen, T. R., Ebrahimi-Fardooee, M. A., van Sorge, N. M., Schneewind, O., Doran, K. S. (2009). Penetration of the Blood-Brain Barrier by Bacillus anthracis Requires the pXO1-Encoded BslA Protein. J. Bacteriol. 191: 7165-7173 [Abstract] [Full Text]  
  • van Schaik, W., Chateau, A., Dillies, M.-A., Coppee, J.-Y., Sonenshein, A. L., Fouet, A. (2009). The Global Regulator CodY Regulates Toxin Gene Expression in Bacillus anthracis and Is Required for Full Virulence. Infect. Immun. 77: 4437-4445 [Abstract] [Full Text]  
  • Pomerantsev, A. P., Camp, A., Leppla, S. H. (2009). A New Minimal Replicon of Bacillus anthracis Plasmid pXO1. J. Bacteriol. 191: 5134-5146 [Abstract] [Full Text]  
  • Hu, X., Van der Auwera, G., Timmery, S., Zhu, L., Mahillon, J. (2009). Distribution, Diversity, and Potential Mobility of Extrachromosomal Elements Related to the Bacillus anthracis pXO1 and pXO2 Virulence Plasmids. Appl. Environ. Microbiol. 75: 3016-3028 [Abstract] [Full Text]  
  • Chand, H. S., Drysdale, M., Lovchik, J., Koehler, T. M., Lipscomb, M. F., Lyons, C. R. (2009). Discriminating Virulence Mechanisms among Bacillus anthracis Strains by Using a Murine Subcutaneous Infection Model. Infect. Immun. 77: 429-435 [Abstract] [Full Text]  
  • Suzuki, H., Sota, M., Brown, C. J., Top, E. M. (2008). Using Mahalanobis distance to compare genomic signatures between bacterial plasmids and chromosomes. Nucleic Acids Res 36: e147-e147 [Abstract] [Full Text]  
  • Leoff, C., Choudhury, B., Saile, E., Quinn, C. P., Carlson, R. W., Kannenberg, E. L. (2008). Structural Elucidation of the Nonclassical Secondary Cell Wall Polysaccharide from Bacillus cereus ATCC 10987: COMPARISON WITH THE POLYSACCHARIDES FROM BACILLUS ANTHRACIS AND B. CEREUS TYPE STRAIN ATCC 14579 REVEALS BOTH UNIQUE AND COMMON STRUCTURAL FEATURES. J. Biol. Chem. 283: 29812-29821 [Abstract] [Full Text]  
  • Bongiorni, C., Fukushima, T., Wilson, A. C., Chiang, C., Mansilla, M. C., Hoch, J. A., Perego, M. (2008). Dual Promoters Control Expression of the Bacillus anthracis Virulence Factor AtxA. J. Bacteriol. 190: 6483-6492 [Abstract] [Full Text]  
  • Dauphin, L. A., Newton, B. R., Rasmussen, M. V., Meyer, R. F., Bowen, M. D. (2008). Gamma Irradiation Can Be Used To Inactivate Bacillus anthracis Spores without Compromising the Sensitivity of Diagnostic Assays. Appl. Environ. Microbiol. 74: 4427-4433 [Abstract] [Full Text]  
  • Hu, X., Fan, W., Han, B., Liu, H., Zheng, D., Li, Q., Dong, W., Yan, J., Gao, M., Berry, C., Yuan, Z. (2008). Complete Genome Sequence of the Mosquitocidal Bacterium Bacillus sphaericus C3-41 and Comparison with Those of Closely Related Bacillus Species. J. Bacteriol. 190: 2892-2902 [Abstract] [Full Text]  
  • Tinsley, E., Khan, S. A. (2007). A Bacillus anthracis-Based In Vitro System Supports Replication of Plasmid pXO2 as Well as Rolling-Circle-Replicating Plasmids. Appl. Environ. Microbiol. 73: 5005-5010 [Abstract] [Full Text]  
  • Sue, D., Marston, C. K., Hoffmaster, A. R., Wilkins, P. P. (2007). Genetic Diversity in a Bacillus anthracis Historical Collection (1954 to 1988). J. Clin. Microbiol. 45: 1777-1782 [Abstract] [Full Text]  
  • Li, J., Miyamoto, K., McClane, B. A. (2007). Comparison of Virulence Plasmids among Clostridium perfringens Type E Isolates. Infect. Immun. 75: 1811-1819 [Abstract] [Full Text]  
  • Rasko, D. A., Rosovitz, M. J., Okstad, O. A., Fouts, D. E., Jiang, L., Cer, R. Z., Kolsto, A.-B., Gill, S. R., Ravel, J. (2007). 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. J. Bacteriol. 189: 52-64 [Abstract] [Full Text]  
  • Van Ert, M. N., Easterday, W. R., Simonson, T. S., U'Ren, J. M., Pearson, T., Kenefic, L. J., Busch, J. D., Huynh, L. Y., Dukerich, M., Trim, C. B., Beaudry, J., Welty-Bernard, A., Read, T., Fraser, C. M., Ravel, J., Keim, P. (2007). Strain-Specific Single-Nucleotide Polymorphism Assays for the Bacillus anthracis Ames Strain. J. Clin. Microbiol. 45: 47-53 [Abstract] [Full Text]  
  • Tang, M., Bideshi, D. K., Park, H.-W., Federici, B. A. (2006). Minireplicon from pBtoxis of Bacillus thuringiensis subsp. israelensis. Appl. Environ. Microbiol. 72: 6948-6954 [Abstract] [Full Text]  
  • Wang, W., Mulakala, C., Ward, S. C., Jung, G., Luong, H., Pham, D., Waring, A. J., Kaznessis, Y., Lu, W., Bradley, K. A., Lehrer, R. I. (2006). Retrocyclins Kill Bacilli and Germinating Spores of Bacillus anthracis and Inactivate Anthrax Lethal Toxin. J. Biol. Chem. 281: 32755-32764 [Abstract] [Full Text]  
  • de Been, M., Francke, C., Moezelaar, R., Abee, T., Siezen, R. J. (2006). Comparative analysis of two-component signal transduction systems of Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis.. Microbiology 152: 3035-3048 [Abstract] [Full Text]  
  • Enkhtuya, J., Kawamoto, K., Kobayashi, Y., Uchida, I., Rana, N., Makino, S.-i. (2006). Significant passive protective effect against anthrax by antibody to Bacillus anthracis inactivated spores that lack two virulence plasmids.. Microbiology 152: 3103-3110 [Abstract] [Full Text]  
  • Hoffmaster, A. R., Hill, K. K., Gee, J. E., Marston, C. K., De, B. K., Popovic, T., Sue, D., Wilkins, P. P., Avashia, S. B., Drumgoole, R., Helma, C. H., Ticknor, L. O., Okinaka, R. T., Jackson, P. J. (2006). Characterization of Bacillus cereus Isolates Associated with Fatal Pneumonias: Strains Are Closely Related to Bacillus anthracis and Harbor B. anthracis Virulence Genes.. J. Clin. Microbiol. 44: 3352-3360 [Abstract] [Full Text]  
  • Shih, Y.-L., Rothfield, L. (2006). The Bacterial Cytoskeleton. Microbiol. Mol. Biol. Rev. 70: 729-754 [Abstract] [Full Text]  
  • White, A. K., Hoch, J. A., Grynberg, M., Godzik, A., Perego, M. (2006). Sensor Domains Encoded in Bacillus anthracis Virulence Plasmids Prevent Sporulation by Hijacking a Sporulation Sensor Histidine Kinase.. J. Bacteriol. 188: 6354-6360 [Abstract] [Full Text]  
  • Klee, S. R., Ozel, M., Appel, B., Boesch, C., Ellerbrok, H., Jacob, D., Holland, G., Leendertz, F. H., Pauli, G., Grunow, R., Nattermann, H. (2006). Characterization of Bacillus anthracis-Like Bacteria Isolated from Wild Great Apes from Cote d'Ivoire and Cameroon.. J. Bacteriol. 188: 5333-5344 [Abstract] [Full Text]  
  • Heffernan, B. J., Thomason, B., Herring-Palmer, A., Shaughnessy, L., McDonald, R., Fisher, N., Huffnagle, G. B., Hanna, P. (2006). Bacillus anthracis Phospholipases C Facilitate Macrophage-Associated Growth and Contribute to Virulence in a Murine Model of Inhalation Anthrax. Infect. Immun. 74: 3756-3764 [Abstract] [Full Text]  
  • Gat, O., Grosfeld, H., Ariel, N., Inbar, I., Zaide, G., Broder, Y., Zvi, A., Chitlaru, T., Altboum, Z., Stein, D., Cohen, S., Shafferman, A. (2006). Search for Bacillus anthracis Potential Vaccine Candidates by a Functional Genomic-Serologic Screen. Infect. Immun. 74: 3987-4001 [Abstract] [Full Text]  
  • Han, C. S., Xie, G., Challacombe, J. F., Altherr, M. R., Bhotika, S. S., Bruce, D., Campbell, C. S., Campbell, M. L., Chen, J., Chertkov, O., Cleland, C., Dimitrijevic, M., Doggett, N. A., Fawcett, J. J., Glavina, T., Goodwin, L. A., Hill, K. K., Hitchcock, P., Jackson, P. J., Keim, P., Kewalramani, A. R., Longmire, J., Lucas, S., Malfatti, S., McMurry, K., Meincke, L. J., Misra, M., Moseman, B. L., Mundt, M., Munk, A. C., Okinaka, R. T., Parson-Quintana, B., Reilly, L. P., Richardson, P., Robinson, D. L., Rubin, E., Saunders, E., Tapia, R., Tesmer, J. G., Thayer, N., Thompson, L. S., Tice, H., Ticknor, L. O., Wills, P. L., Brettin, T. S., Gilna, P. (2006). Pathogenomic Sequence Analysis of Bacillus cereus and Bacillus thuringiensis Isolates Closely Related to Bacillus anthracis. J. Bacteriol. 188: 3382-3390 [Abstract] [Full Text]  
  • Mikosa, M., Sochacka-Pietal, M., Baj, J., Bartosik, D. (2006). Identification of a transposable genomic island of Paracoccus pantotrophus DSM 11072 by its transposition to a novel entrapment vector pMMB2.. Microbiology 152: 1063-1073 [Abstract] [Full Text]  
  • Stratilo, C. W., Lewis, C. T., Bryden, L., Mulvey, M. R., Bader, D. (2006). Single-Nucleotide Repeat Analysis for Subtyping Bacillus anthracis Isolates.. J. Clin. Microbiol. 44: 777-782 [Abstract] [Full Text]  
  • Bongiorni, C., Stoessel, R., Shoemaker, D., Perego, M. (2006). Rap Phosphatase of Virulence Plasmid pXO1 Inhibits Bacillus anthracis Sporulation. J. Bacteriol. 188: 487-498 [Abstract] [Full Text]  
  • Tourasse, N. J., Stabell, F. B., Reiter, L., Kolsto, A.-B. (2005). Unusual Group II Introns in Bacteria of the Bacillus cereus Group. J. Bacteriol. 187: 5437-5451 [Abstract] [Full Text]  
  • Waller, L. N., Stump, M. J., Fox, K. F., Harley, W. M., Fox, A., Stewart, G. C., Shahgholi, M. (2005). Identification of a Second Collagen-Like Glycoprotein Produced by Bacillus anthracis and Demonstration of Associated Spore-Specific Sugars. J. Bacteriol. 187: 4592-4597 [Abstract] [Full Text]  
  • Gaspar, A. H., Marraffini, L. A., Glass, E. M., DeBord, K. L., Ton-That, H., Schneewind, O. (2005). Bacillus anthracis Sortase A (SrtA) Anchors LPXTG Motif-Containing Surface Proteins to the Cell Wall Envelope. J. Bacteriol. 187: 4646-4655 [Abstract] [Full Text]  
  • Kunnimalaiyaan, M., Vary, P. S. (2005). Molecular Characterization of Plasmid pBM300 from Bacillus megaterium QM B1551. Appl. Environ. Microbiol. 71: 3068-3076 [Abstract] [Full Text]  
  • Aronson, A. I., Bell, C., Fulroth, B. (2005). Plasmid-Encoded Regulator of Extracellular Proteases in Bacillus anthracis. J. Bacteriol. 187: 3133-3138 [Abstract] [Full Text]  
  • Stinear, T. P., Pryor, M. J., Porter, J. L., Cole, S. T. (2005). Functional analysis and annotation of the virulence plasmid pMUM001 from Mycobacterium ulcerans. Microbiology 151: 683-692 [Abstract] [Full Text]  
  • Barbosa, T. M., Serra, C. R., La Ragione, R. M., Woodward, M. J., Henriques, A. O. (2005). Screening for Bacillus Isolates in the Broiler Gastrointestinal Tract. Appl. Environ. Microbiol. 71: 968-978 [Abstract] [Full Text]  
  • Phipps, A. J., Premanandan, C., Barnewall, R. E., Lairmore, M. D. (2004). Rabbit and Nonhuman Primate Models of Toxin-Targeting Human Anthrax Vaccines. Microbiol. Mol. Biol. Rev. 68: 617-629 [Abstract] [Full Text]  
  • Okstad, O. A., Tourasse, N. J., Stabell, F. B., Sundfaer, C. K., Egge-Jacobsen, W., Risoen, P. A., Read, T. D., Kolsto, A.-B. (2004). The bcr1 DNA Repeat Element Is Specific to the Bacillus cereus Group and Exhibits Mobile Element Characteristics. J. Bacteriol. 186: 7714-7725 [Abstract] [Full Text]  
  • Barth, H., Aktories, K., Popoff, M. R., Stiles, B. G. (2004). Binary Bacterial Toxins: Biochemistry, Biology, and Applications of Common Clostridium and Bacillus Proteins. Microbiol. Mol. Biol. Rev. 68: 373-402 [Abstract] [Full Text]  
  • Reissbrodt, R., Rassbach, A., Burghardt, B., Rienacker, I., Mietke, H., Schleif, J., Tschape, H., Lyte, M., Williams, P. H. (2004). Assessment of a New Selective Chromogenic Bacillus cereus Group Plating Medium and Use of Enterobacterial Autoinducer of Growth for Cultural Identification of Bacillus Species. J. Clin. Microbiol. 42: 3795-3798 [Abstract] [Full Text]  
  • Hoffmaster, A. R., Ravel, J., Rasko, D. A., Chapman, G. D., Chute, M. D., Marston, C. K., De, B. K., Sacchi, C. T., Fitzgerald, C., Mayer, L. W., Maiden, M. C. J., Priest, F. G., Barker, M., Jiang, L., Cer, R. Z., Rilstone, J., Peterson, S. N., Weyant, R. S., Galloway, D. R., Read, T. D., Popovic, T., Fraser, C. M. (2004). Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax. Proc. Natl. Acad. Sci. USA 101: 8449-8454 [Abstract] [Full Text]  
  • Rasko, D. A., Ravel, J., Okstad, O. A., Helgason, E., Cer, R. Z., Jiang, L., Shores, K. A., Fouts, D. E., Tourasse, N. J., Angiuoli, S. V., Kolonay, J., Nelson, W. C., Kolsto, A.-B., Fraser, C. M., Read, T. D. (2004). The genome sequence of Bacillus cereus ATCC 10987 reveals metabolic adaptations and a large plasmid related to Bacillus anthracis pXO1. Nucleic Acids Res 32: 977-988 [Abstract] [Full Text]  
  • Zhang, R., Zhang, C.-T. (2003). Identification of genomic islands in the genome of Bacillus cereus by comparative analysis with Bacillus anthracis. Physiol. Genomics 16: 19-23 [Abstract] [Full Text]  
  • Scholle, M. D., White, C. A., Kunnimalaiyaan, M., Vary, P. S. (2003). Sequencing and Characterization of pBM400 from Bacillus megaterium QM B1551. Appl. Environ. Microbiol. 69: 6888-6898 [Abstract] [Full Text]  
  • Pomerantsev, A. P., Kalnin, K. V., Osorio, M., Leppla, S. H. (2003). Phosphatidylcholine-Specific Phospholipase C and Sphingomyelinase Activities in Bacteria of the Bacillus cereus Group. Infect. Immun. 71: 6591-6606 [Abstract] [Full Text]  
  • Grohmann, E., Muth, G., Espinosa, M. (2003). Conjugative Plasmid Transfer in Gram-Positive Bacteria. Microbiol. Mol. Biol. Rev. 67: 277-301 [Abstract] [Full Text]  
  • Bourgogne, A., Drysdale, M., Hilsenbeck, S. G., Peterson, S. N., Koehler, T. M. (2003). Global Effects of Virulence Gene Regulators in a Bacillus anthracis Strain with Both Virulence Plasmids. Infect. Immun. 71: 2736-2743 [Abstract] [Full Text]  
  • Chen, Y., Succi, J., Tenover, F. C., Koehler, T. M. (2003). {beta}-Lactamase Genes of the Penicillin-Susceptible Bacillus anthracis Sterne Strain. J. Bacteriol. 185: 823-830 [Abstract] [Full Text]  
  • Fasanella, A., Losito, S., Adone, R., Ciuchini, F., Trotta, T., Altamura, S. A., Chiocco, D., Ippolito, G. (2003). PCR Assay To Detect Bacillus anthracis Spores in Heat-Treated Specimens. J. Clin. Microbiol. 41: 896-899 [Abstract] [Full Text]  
  • Ariel, N., Zvi, A., Grosfeld, H., Gat, O., Inbar, Y., Velan, B., Cohen, S., Shafferman, A. (2002). Search for Potential Vaccine Candidate Open Reading Frames in the Bacillus anthracis Virulence Plasmid pXO1: In Silico and In Vitro Screening. Infect. Immun. 70: 6817-6827 [Abstract] [Full Text]  
  • Puskarova, A., Ferianc, P., Kormanec, J., Homerova, D., Farewell, A., Nystrom, T. (2002). Regulation of yodA encoding a novel cadmium-induced protein in Escherichia coli. Microbiology 148: 3801-3811 [Abstract] [Full Text]  
  • Berry, C., O'Neil, S., Ben-Dov, E., Jones, A. F., Murphy, L., Quail, M. A., Holden, M. T. G., Harris, D., Zaritsky, A., Parkhill, J. (2002). Complete Sequence and Organization of pBtoxis, the Toxin-Coding Plasmid of Bacillus thuringiensis subsp. israelensis. Appl. Environ. Microbiol. 68: 5082-5095 [Abstract] [Full Text]  
  • Read, T. D., Salzberg, S. L., Pop, M., Shumway, M., Umayam, L., Jiang, L., Holtzapple, E., Busch, J. D., Smith, K. L., Schupp, J. M., Solomon, D., Keim, P., Fraser, C. M. (2002). Comparative Genome Sequencing for Discovery of Novel Polymorphisms in Bacillus anthracis. Science 296: 2028-2033 [Abstract] [Full Text]  
  • Saile, E., Koehler, T. M. (2002). Control of Anthrax Toxin Gene Expression by the Transition State Regulator abrB. J. Bacteriol. 184: 370-380 [Abstract] [Full Text]  
  • Ticknor, L. O., Kolsto, A.-B., Hill, K. K., Keim, P., Laker, M. T., Tonks, M., Jackson, P. J. (2001). Fluorescent Amplified Fragment Length Polymorphism Analysis of Norwegian Bacillus cereus and Bacillus thuringiensis Soil Isolates. Appl. Environ. Microbiol. 67: 4863-4873 [Abstract] [Full Text]  
  • Martinez, B., Tomkins, J., Wackett, L. P., Wing, R., Sadowsky, M. J. (2001). Complete Nucleotide Sequence and Organization of the Atrazine Catabolic Plasmid pADP-1 from Pseudomonas sp. Strain ADP. J. Bacteriol. 183: 5684-5697 [Abstract] [Full Text]  
  • Qi, Y., Patra, G., Liang, X., Williams, L. E., Rose, S., Redkar, R. J., DelVecchio, V. G. (2001). Utilization of the rpoB Gene as a Specific Chromosomal Marker for Real-Time PCR Detection of Bacillus anthracis. Appl. Environ. Microbiol. 67: 3720-3727 [Abstract] [Full Text]  
  • Lewis, J. A., Hatfull, G. F. (2001). Control of directionality in integrase-mediated recombination: examination of recombination directionality factors (RDFs) including Xis and Cox proteins. Nucleic Acids Res 29: 2205-2216 [Abstract] [Full Text]  
  • Østergaard, S., Brøndsted, L., Vogensen, F. K. (2001). Identification of a Replication Protein and Repeats Essential for DNA Replication of the Temperate Lactococcal Bacteriophage TP901-1. Appl. Environ. Microbiol. 67: 774-781 [Abstract] [Full Text]  
  • Keim, P., Price, L. B., Klevytska, A. M., Smith, K. L., Schupp, J. M., Okinaka, R., Jackson, P. J., Hugh-Jones, M. E. (2000). Multiple-Locus Variable-Number Tandem Repeat Analysis Reveals Genetic Relationships within Bacillus anthracis. J. Bacteriol. 182: 2928-2936 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Okinaka, R. T.
Right arrow Articles by Jackson, P. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Okinaka, R. T.
Right arrow Articles by Jackson, P. J.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»