The Bacillus anthracis Carbosap genome, which includes the pXO1 and pXO2 plasmids, has been shown to encode the major B. anthracis virulence factors, yet this strain’s attenuation has not yet been explained. Here we report the draft genome sequence of this strain, and a comparison to fully virulent B. anthracis

Bergman, Nicholas H.

Blank, Thomas E.

Burans, James P.

Cendrowski, Stephen R.

Diviak, Lynn

Friedlander, Arthur M.

Friss, Mary Beth

Harrington, Robin

Hnath, Jonathan

Karaolis, David

Klubnik, Joy

Ondov, Brian D.

Phillippy, Adam M.

Radune, Diana

Rosovitz, M. J.

Treangen, Todd

English

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln US Army Research U.S. Department of Defense 2013 Genome Sequence of the Attenuated Carbosap Vaccine Strain of Bacillus anthracis Robin Harrington National Biodefense Analysis and Countermeasures Center Brian D. Ondov National Biodefense Analysis and Countermeasures Center Diana Radune National Biodefense Analysis and Countermeasures Center Mary Beth Friss National Biodefense Analysis and Countermeasures Center Joy Klubnik National Biodefense Analysis and Countermeasures Center See next page for additional authors Follow this and additional works at: https://digitalcommons.unl.edu/usarmyresearch Harrington, Robin; Ondov, Brian D.; Radune, Diana; Friss, Mary Beth; Klubnik, Joy; Diviak, Lynn; Hnath, Jonathan; Cendrowski, Stephen R.; Blank, Thomas E.; Karaolis, David; Friedlander, Arthur M.; Burans, James P.; Rosovitz, M. J.; Treangen, Todd; Phillippy, Adam M.; and Bergman, Nicholas H., "Genome Sequence of the Attenuated Carbosap Vaccine Strain of Bacillus anthracis" (2013). US Army Research. 241. https://digitalcommons.unl.edu/usarmyresearch/241 This Article is brought to you for free and open access by the U.S. Department of Defense at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in US Army Research by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors Robin Harrington, Brian D. Ondov, Diana Radune, Mary Beth Friss, Joy Klubnik, Lynn Diviak, Jonathan Hnath, Stephen R. Cendrowski, Thomas E. Blank, David Karaolis, Arthur M. Friedlander, James P. Burans, M. J. Rosovitz, Todd Treangen, Adam M. Phillippy, and Nicholas H. Bergman This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/usarmyresearch/241 Genome Sequence of the Attenuated Carbosap Vaccine Strain ofBacillus anthracisRobin Harrington,a Brian D. Ondov,a Diana Radune,a Mary Beth Friss,a Joy Klubnik,a Lynn Diviak,a Jonathan Hnath,aStephen R. Cendrowski,a Thomas E. Blank,a David Karaolis,a Arthur M. Friedlander,b James P. Burans,a M. J. Rosovitz,aTodd Treangen,a Adam M. Phillippy,a Nicholas H. BergmanaNational Biodefense Analysis and Countermeasures Center, Frederick, Maryland, USAa; United States Army Medical Research Institute for Infectious Diseases, Frederick,Maryland, USAbThe Bacillus anthracis Carbosap genome, which includes the pXO1 and pXO2 plasmids, has been shown to encode the majorB. anthracis virulence factors, yet this strain’s attenuation has not yet been explained. Here we report the draft genome sequenceof this strain, and a comparison to fully virulent B. anthracis.Received 23 October 2012 Accepted 7 November 2012 Published 15 January 2013Citation Harrington R, Ondov BD, Radune D, Friss MB, Klubnik J, Diviak L, Hnath J, Cendrowski SR, Blank TE, Karaolis D, Friedlander AM, Burans JP, Rosovitz MJ, Treangen T,Phillippy AM, Bergman NH. 2013. Genome sequence of the attenuated Carbosap vaccine strain of Bacillus anthracis. Genome Announc. 1(1):e00067-12. doi:10.1128/genomeA.00067-12.Copyright © 2013 Harrington et al. This is an open-access article distributed under the terms of the Attribution 3.0 Unported Creative Commons License.Address correspondence to Nicholas H. Bergman, nicholas.bergman@nbacc.dhs.gov.Bacillus anthracis is a Gram-positive, spore-forming, filamen-tous bacillus and is the causative agent of anthrax in bothanimals and humans (1). B. anthracis strains attenuated for viru-lence in animals that were described in the latter part of the 19thcentury, and in particular, those put forward by Pasteur, sooncame into use for vaccination against anthrax in cattle and sheep(2).Many attenuated strains of B. anthracis lack one of the twovirulence-associated plasmids pXO1 and pXO2. The Sterne-typestrains lack pXO2 and are toxigenic but cannot produce a capsule,while the Pasteur-type vaccine strains lack the pXO1 plasmid andare therefore encapsulated but nontoxigenic. Less commonly,other strains have been shown to be attenuated despite retainingboth the pXO1 and pXO2 plasmids, which would normally resultin full virulence (3, 4).One example of such a strain is B. anthracis Carbosap, used inItaly as a live spore vaccine for cattle and sheep (5). The Carbosapgenome has been previously shown to contain sequences that in-dicate the presence of pXO1 and pXO2 as well as a limited set ofchromosomal markers associated with virulence, and yet it showssignificant attenuation of virulence in rabbits (5, 6, 7).In order to define the differences between Carbosap and otherstrains of B. anthracis that may explain its attenuation, we se-quenced the genome of Carbosap. Genomic DNA was preparedfrom the Carbosap strain of B. anthracis (obtained from A.Fasanella) using standard methods (8) and sequenced using the454 and Illumina MiSeq platforms. A total of 344,312 454 readsand 1,719,544 Illumina reads, totaling 390 Mb, were assembledusing Newbler version 2.7 in both de novo and reference modes,using B. anthracis strain Ames Ancestor as a reference (9). Contigsfrom the de novo assembly that showed structural variation withrespect to the reference were combined with the reference assem-bly using Minimus 2 (part of AMOS 3.1.0) to produce a draftgenome of 5,402,970 bp and 21 contigs.Whole-genome single-nucleotide polymorphism (SNP) anal-ysis showed that the Carbosap strain is a member of the trans-Eurasian clade of B. anthracis; the genome also contains 3 chro-mosomal deletions of 29, 24, and 3.5 kb relative to virulent strainsof B. anthracis, consistent with previous array and PCR analyses(10). There were no major changes found in the Carbosap pXO1and pXO2 sequences relative to plasmids from the Ames Ancestorstrain. The chromosomal deletions contain 50 annotated genes,many of which have a known or proposed function that could belinked to virulence, so the precise nature of Carbosap’s attenua-tion remains unclear. A similar situation was recently reported forthe B. anthracis CDC 684 strain (11), and it seems likely that inboth cases further investigation into the functional effects of thegenomic changes in these two strains will yield new insights intoB. anthracis pathogenesis and aid in the identification of potentialtargets for development of new anthrax therapeutics.Nucleotide sequence accession number. This Whole GenomeShotgun project has been deposited at DDBJ/EMBL/GenBank un-der the accession no. ANAO00000000. The version described inthis paper is the first version.ACKNOWLEDGMENTSThis work was funded under Agreement No. HSHQDC-07-C-00020awarded to the Battelle National Biodefense Institute (BNBI) by the De-partment of Homeland Security (DHS) for the management and opera-tion of the National Biodefense Analysis and Countermeasures Center(NBACC), a Federally Funded Research and Development Center.The views and conclusions contained in this document are those of theauthors and should not be interpreted as necessarily representing the of-ficial policies, either expressed or implied, of the U.S. Department ofHomeland Security or the United States Government.REFERENCES1. Sweeney DA, Hicks CW, Cui X, Li Y, Eichacker PQ. 2011. Anthraxinfection. Am. J. Respir. Crit. Care Med. 184(12):1333–1341.2. Turnbull PC. 1991. Anthrax vaccines: past, present and future. Vaccine9(8):533–539.Genome AnnouncementsJanuary/February 2013 Volume 1 Issue 1 e00067-12 genomea.asm.org 13. Mikesell P, Ivins BE, Ristroph JD, Dreier TM. 1983. Evidence forplasmid-mediated toxin production in Bacillus anthracis. Infect. Immun.39(1):371–376.4. Uchida I, Sekizaki T, Hashimoto K, Terakado N. 1985. Association ofthe encapsulation of Bacillus anthracis with a 60 megadalton plasmid. J.Gen. Microbiol. 131(2):363–367.5. Fasanella A, Losito S, Trotta T, Adone R, Massa S, Ciuchini F, ChioccoD. 2001. Detection of anthrax vaccine virulence factors by polymerasechain reaction. Vaccine 19(30):4214 – 4218.6. Adone R, Pasquali P, La Rosa G, Marianelli C, Muscillo M, Fasanella A,Francia M, Ciuchini F. 2002. Sequence analysis of the genes encoding forthe major virulence factors of Bacillus anthracis vaccine strain “Carbosap”.J. Appl. Microbiol. 93(1):117–121.7. La Rosa G, Muscillo M, Sali M, De Carolis E, Marianelli C, Ciuchini F,Fasanella A, Adone R. 2006. Molecular study of the genes involved invirulence regulatory pathways in Bacillus anthracis vaccine strain “Car-bosap”. New Microbiol. 29(4):307–310.8. Goldenberger D, Perschil I, Ritzler M, Altwegg M. 1995. A simple“universal” DNA extraction procedure using SDS and proteinase K iscompatible with direct PCR amplification. PCR Methods Appl. 4(6):368 –370.9. Ravel J, Jiang L, Stanley ST, Wilson MR, Decker RS, Read TD, Wor-sham P, Keim PS, Salzberg SL, Fraser-Liggett CM, Rasko DA. 2009. Thecomplete genome sequence of Bacillus anthracis Ames Ancestor. J. Bacte-riol. 191(1):445– 446.10. Blank TE, Rasmussen SL, Gajer P, Carpenter BM, Chabot DJ, RaskoDA, Ravel J, Peterson SN, Friedlander AM. 2005. The Carbosap vaccinestrain of Bacillus anthracis contains chromosomal deletions, abstr P115, p74. Abstr. Bacillus ACT 2005 Conference, Santa Fe, New Mexico.11. Okinaka RT, Price EP, Wolken SR, Gruendike JM, Chung WK, PearsonT, Xie G, Munk C, Hill KK, Challacombe J, Ivins BE, Schupp JM,Beckstrom-Sternberg SM, Friedlander A, Keim P. 2011. An attenuatedstrain of Bacillus anthracis (CDC 684) has a large chromosomal inversionand altered growth kinetics. BMC Genomics 12:477.Harrington et al.Genome Announcements2 genomea.asm.org January/February 2013 Volume 1 Issue 1 e00067-12

Genome Sequence of the Attenuated Carbosap Vaccine Strain of
\u3ci\u3eBacillus anthracis\u3c/i\u3e

UNL | Libraries

University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
US Army Research U.S. Department of Defense 
2013 
Genome Sequence of the Attenuated Carbosap Vaccine Strain of 
Bacillus anthracis 
Robin Harrington 
National Biodefense Analysis and Countermeasures Center 
Brian D. Ondov 
National Biodefense Analysis and Countermeasures Center 
Diana Radune 
National Biodefense Analysis and Countermeasures Center 
Mary Beth Friss 
National Biodefense Analysis and Countermeasures Center 
Joy Klubnik 
National Biodefense Analysis and Countermeasures Center 
See next page for additional authors 
Follow this and additional works at: https://digitalcommons.unl.edu/usarmyresearch 
Harrington, Robin; Ondov, Brian D.; Radune, Diana; Friss, Mary Beth; Klubnik, Joy; Diviak, Lynn; Hnath, 
Jonathan; Cendrowski, Stephen R.; Blank, Thomas E.; Karaolis, David; Friedlander, Arthur M.; Burans, 
James P.; Rosovitz, M. J.; Treangen, Todd; Phillippy, Adam M.; and Bergman, Nicholas H., "Genome 
Sequence of the Attenuated Carbosap Vaccine Strain of Bacillus anthracis" (2013). US Army Research. 
241. 
https://digitalcommons.unl.edu/usarmyresearch/241 
This Article is brought to you for free and open access by the U.S. Department of Defense at 
DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in US Army Research by an 
authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 
Authors 
Robin Harrington, Brian D. Ondov, Diana Radune, Mary Beth Friss, Joy Klubnik, Lynn Diviak, Jonathan 
Hnath, Stephen R. Cendrowski, Thomas E. Blank, David Karaolis, Arthur M. Friedlander, James P. Burans, 
M. J. Rosovitz, Todd Treangen, Adam M. Phillippy, and Nicholas H. Bergman 
This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/
usarmyresearch/241 
Genome Sequence of the Attenuated Carbosap Vaccine Strain of
Bacillus anthracis
Robin Harrington,a Brian D. Ondov,a Diana Radune,a Mary Beth Friss,a Joy Klubnik,a Lynn Diviak,a Jonathan Hnath,a
Stephen R. Cendrowski,a Thomas E. Blank,a David Karaolis,a Arthur M. Friedlander,b James P. Burans,a M. J. Rosovitz,a
Todd Treangen,a Adam M. Phillippy,a Nicholas H. Bergmana
National Biodefense Analysis and Countermeasures Center, Frederick, Maryland, USAa; United States Army Medical Research Institute for Infectious Diseases, Frederick,
Maryland, USAb
The Bacillus anthracis Carbosap genome, which includes the pXO1 and pXO2 plasmids, has been shown to encode the major
B. anthracis virulence factors, yet this strain’s attenuation has not yet been explained. Here we report the draft genome sequence
of this strain, and a comparison to fully virulent B. anthracis.
Received 23 October 2012 Accepted 7 November 2012 Published 15 January 2013
Citation Harrington R, Ondov BD, Radune D, Friss MB, Klubnik J, Diviak L, Hnath J, Cendrowski SR, Blank TE, Karaolis D, Friedlander AM, Burans JP, Rosovitz MJ, Treangen T,
Phillippy AM, Bergman NH. 2013. Genome sequence of the attenuated Carbosap vaccine strain of Bacillus anthracis. Genome Announc. 1(1):e00067-12. doi:10.1128/
genomeA.00067-12.
Copyright © 2013 Harrington et al. This is an open-access article distributed under the terms of the Attribution 3.0 Unported Creative Commons License.
Address correspondence to Nicholas H. Bergman, nicholas.bergman@nbacc.dhs.gov.
Bacillus anthracis is a Gram-positive, spore-forming, filamen-tous bacillus and is the causative agent of anthrax in both
animals and humans (1). B. anthracis strains attenuated for viru-
lence in animals that were described in the latter part of the 19th
century, and in particular, those put forward by Pasteur, soon
came into use for vaccination against anthrax in cattle and sheep
(2).
Many attenuated strains of B. anthracis lack one of the two
virulence-associated plasmids pXO1 and pXO2. The Sterne-type
strains lack pXO2 and are toxigenic but cannot produce a capsule,
while the Pasteur-type vaccine strains lack the pXO1 plasmid and
are therefore encapsulated but nontoxigenic. Less commonly,
other strains have been shown to be attenuated despite retaining
both the pXO1 and pXO2 plasmids, which would normally result
in full virulence (3, 4).
One example of such a strain is B. anthracis Carbosap, used in
Italy as a live spore vaccine for cattle and sheep (5). The Carbosap
genome has been previously shown to contain sequences that in-
dicate the presence of pXO1 and pXO2 as well as a limited set of
chromosomal markers associated with virulence, and yet it shows
significant attenuation of virulence in rabbits (5, 6, 7).
In order to define the differences between Carbosap and other
strains of B. anthracis that may explain its attenuation, we se-
quenced the genome of Carbosap. Genomic DNA was prepared
from the Carbosap strain of B. anthracis (obtained from A.
Fasanella) using standard methods (8) and sequenced using the
454 and Illumina MiSeq platforms. A total of 344,312 454 reads
and 1,719,544 Illumina reads, totaling 390 Mb, were assembled
using Newbler version 2.7 in both de novo and reference modes,
using B. anthracis strain Ames Ancestor as a reference (9). Contigs
from the de novo assembly that showed structural variation with
respect to the reference were combined with the reference assem-
bly using Minimus 2 (part of AMOS 3.1.0) to produce a draft
genome of 5,402,970 bp and 21 contigs.
Whole-genome single-nucleotide polymorphism (SNP) anal-
ysis showed that the Carbosap strain is a member of the trans-
Eurasian clade of B. anthracis; the genome also contains 3 chro-
mosomal deletions of 29, 24, and 3.5 kb relative to virulent strains
of B. anthracis, consistent with previous array and PCR analyses
(10). There were no major changes found in the Carbosap pXO1
and pXO2 sequences relative to plasmids from the Ames Ancestor
strain. The chromosomal deletions contain 50 annotated genes,
many of which have a known or proposed function that could be
linked to virulence, so the precise nature of Carbosap’s attenua-
tion remains unclear. A similar situation was recently reported for
the B. anthracis CDC 684 strain (11), and it seems likely that in
both cases further investigation into the functional effects of the
genomic changes in these two strains will yield new insights into
B. anthracis pathogenesis and aid in the identification of potential
targets for development of new anthrax therapeutics.
Nucleotide sequence accession number. This Whole Genome
Shotgun project has been deposited at DDBJ/EMBL/GenBank un-
der the accession no. ANAO00000000. The version described in
this paper is the first version.
ACKNOWLEDGMENTS
This work was funded under Agreement No. HSHQDC-07-C-00020
awarded to the Battelle National Biodefense Institute (BNBI) by the De-
partment of Homeland Security (DHS) for the management and opera-
tion of the National Biodefense Analysis and Countermeasures Center
(NBACC), a Federally Funded Research and Development Center.
The views and conclusions contained in this document are those of the
authors and should not be interpreted as necessarily representing the of-
ficial policies, either expressed or implied, of the U.S. Department of
Homeland Security or the United States Government.
REFERENCES
1. Sweeney DA, Hicks CW, Cui X, Li Y, Eichacker PQ. 2011. Anthrax
infection. Am. J. Respir. Crit. Care Med. 184(12):1333–1341.
2. Turnbull PC. 1991. Anthrax vaccines: past, present and future. Vaccine
9(8):533–539.
Genome AnnouncementsJanuary/February 2013 Volume 1 Issue 1 e00067-12 genomea.asm.org 1
3. Mikesell P, Ivins BE, Ristroph JD, Dreier TM. 1983. Evidence for
plasmid-mediated toxin production in Bacillus anthracis. Infect. Immun.
39(1):371–376.
4. Uchida I, Sekizaki T, Hashimoto K, Terakado N. 1985. Association of
the encapsulation of Bacillus anthracis with a 60 megadalton plasmid. J.
Gen. Microbiol. 131(2):363–367.
5. Fasanella A, Losito S, Trotta T, Adone R, Massa S, Ciuchini F, Chiocco
D. 2001. Detection of anthrax vaccine virulence factors by polymerase
chain reaction. Vaccine 19(30):4214 – 4218.
6. Adone R, Pasquali P, La Rosa G, Marianelli C, Muscillo M, Fasanella A,
Francia M, Ciuchini F. 2002. Sequence analysis of the genes encoding for
the major virulence factors of Bacillus anthracis vaccine strain “Carbosap”.
J. Appl. Microbiol. 93(1):117–121.
7. La Rosa G, Muscillo M, Sali M, De Carolis E, Marianelli C, Ciuchini F,
Fasanella A, Adone R. 2006. Molecular study of the genes involved in
virulence regulatory pathways in Bacillus anthracis vaccine strain “Car-
bosap”. New Microbiol. 29(4):307–310.
8. Goldenberger D, Perschil I, Ritzler M, Altwegg M. 1995. A simple
“universal” DNA extraction procedure using SDS and proteinase K is
compatible with direct PCR amplification. PCR Methods Appl. 4(6):
368 –370.
9. Ravel J, Jiang L, Stanley ST, Wilson MR, Decker RS, Read TD, Wor-
sham P, Keim PS, Salzberg SL, Fraser-Liggett CM, Rasko DA. 2009. The
complete genome sequence of Bacillus anthracis Ames Ancestor. J. Bacte-
riol. 191(1):445– 446.
10. Blank TE, Rasmussen SL, Gajer P, Carpenter BM, Chabot DJ, Rasko
DA, Ravel J, Peterson SN, Friedlander AM. 2005. The Carbosap vaccine
strain of Bacillus anthracis contains chromosomal deletions, abstr P115, p
74. Abstr. Bacillus ACT 2005 Conference, Santa Fe, New Mexico.
11. Okinaka RT, Price EP, Wolken SR, Gruendike JM, Chung WK, Pearson
T, Xie G, Munk C, Hill KK, Challacombe J, Ivins BE, Schupp JM,
Beckstrom-Sternberg SM, Friedlander A, Keim P. 2011. An attenuated
strain of Bacillus anthracis (CDC 684) has a large chromosomal inversion
and altered growth kinetics. BMC Genomics 12:477.
Harrington et al.
Genome Announcements2 genomea.asm.org January/February 2013 Volume 1 Issue 1 e00067-12


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Genome Sequence of the Attenuated Carbosap Vaccine Strain of \u3ci\u3eBacillus anthracis\u3c/i\u3e

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