INFECTION AND IMMUNITY, Jan. 2006, p. 682–693
Vol. 74, No. 1
Genome Engineering in Bacillus anthracis Using Cre Recombinase
Andrei P. Pomerantsev, Ramakrishnan Sitaraman,† Craig R. Galloway, Violetta Kivovich,
and Stephen H. Leppla*
Bacterial Toxins and Therapeutics Section, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, Maryland 20892-4349
Received 8 August 2005/Returned for modification 22 September 2005/Accepted 13 October 2005
Genome engineering is a powerful method for the study of bacterial virulence. With the availability of the
complete genomic sequence of Bacillus anthracis, it is now possible to inactivate or delete selected genes of
interest. However, many current methods for disrupting or deleting more than one gene require use of multiple
antibiotic resistance determinants. In this report we used an approach that temporarily inserts an antibiotic
resistance marker into a selected region of the genome and subsequently removes it, leaving the target region
(a single gene or a larger genomic segment) permanently mutated. For this purpose, a spectinomycin resistance
cassette flanked by bacteriophage P1 loxP sites oriented as direct repeats was inserted within a selected gene.
After identification of strains having the spectinomycin cassette inserted by a double-crossover event, a
thermo-sensitive plasmid expressing Cre recombinase was introduced at the permissive temperature. Cre
recombinase action at the loxP sites excised the spectinomycin marker, leaving a single loxP site within the
targeted gene or genomic segment. The Cre-expressing plasmid was then removed by growth at the restrictive
temperature. The procedure could then be repeated to mutate additional genes. In this way, we sequentially
mutated two pairs of genes: pepM and spo0A, and mcrB and mrr. Furthermore, loxP sites introduced at distant
genes could be recombined by Cre recombinase to cause deletion of large intervening regions. In this way, we
deleted the capBCAD region of the pXO2 plasmid and the entire 30 kb of chromosomal DNA between the mcrB
and mrr genes, and in the latter case we found that the 32 intervening open reading frames were not essential
Bacillus anthracis, the etiological agent of anthrax, is a gram-
positive, rod-shaped, spore-forming bacterium. Two major fac-
tors encoded on two large plasmids are associated with viru-
lence. Plasmid pXO2 encodes a poly-?-D-glutamic acid capsule
(10, 19), whereas pXO1 encodes the anthrax toxins, consisting
of protective antigen (PA), lethal factor, and edema factor (10,
16, 19, 22). Vaccination against anthrax is the most successful
way of preventing morbidity and mortality associated with an-
thrax infections. In the United States, persons at risk of expo-
sure receive a licensed anthrax vaccine (BioThrax), which is
aluminum hydroxide-adsorbed, formalin-treated culture su-
pernatant of the attenuated toxigenic, noncapsulated, low-pro-
teolytic B. anthracis strain V770-NP1-R. The key immunogen
in the vaccine is PA (17), but undefined degradation products
as well as other anthrax toxin components are present. Several
approaches have been used to obtain improved anthrax vac-
cines, including expression of PA in Bacillus subtilis (37). The
recombinant PA (rPA) produced by B. subtilis is, however,
rapidly degraded by cell-associated or secreted proteases, re-
sulting in a reduced yield of protein. The current recombinant
PA vaccine under development in the United States is pro-
duced in a sporulation-deficient, virulence plasmid-cured
strain of B. anthracis containing a vector expressing only PA
(8). While effective, this host strain might be improved by
disruption of genes encoding extracellular proteases that con-
tribute to degradation of the secreted PA.
The availability of the B. anthracis genomic sequence (31)
has greatly facilitated the genetic dissection of factors deter-
mining this organism’s fitness and pathogenic ability. Typically,
gene function is assessed by inserting an antibiotic resistance
marker into a target gene so as to disrupt it. However, when it
becomes necessary to target multiple genes, the limited num-
ber of convenient selectable markers makes this difficult. This
problem has been solved in other bacteria by use of site-
specific recombinases. The 38-kDa Cre (causing recombina-
tion) recombinase of bacteriophage P1 recognizes a 34-bp loxP
(locus of crossing over) site which consists of two 13-bp in-
verted repeats surrounding an 8-bp asymmetric core sequence
(34). The Cre-loxP system has previously been employed to
excise antibiotic resistance genes from Escherichia coli after
their introduction into specific target genes. The recombina-
tion reaction results in the excision or inversion of the inter-
vening sequence between two loxP, depending on their relative
orientation (24). In this report we describe the adaptation of
the Cre-loxP system for removing spectinomycin resistance
genes from B. anthracis mutants in a sequential fashion. The
method was first applied to the mutation of two genes, spo0A
and pepM. The spo0A gene encodes a global regulator shown
to be essential for spore formation (9). The pepM gene encodes
a secreted, zinc-dependent metalloprotease (also referred to as
the “neutral protease/peptidase”) belonging to the M4 metal-
lopeptidase family (Protein Families database; Sanger Insti-
tute, United Kingdom). Mutations in spo0A and pepM were
generated individually and sequentially in three attenuated
* Corresponding author. Mailing address: Bacterial Toxins and
Therapeutics Section, National Institute of Allergy and Infectious Dis-
eases, National Institutes of Health, Bethesda, MD 20892-4349.
Phone: (301) 594-2865. Fax: (301) 480-0326. E-mail: firstname.lastname@example.org
† Present address: Department of Medicine, Division of Gastroen-
terology, Vanderbilt University, Nashville, TN 37232.
strains of B. anthracis: pX01?pX02?, pX01?pX02?, and
plasmid free. The plasmid-free, nonsporogenic, low-proteolytic
strain is suggested to have value as a host strain for producing
recombinant PA for use in vaccines.
B. anthracis is refractory to transformation by DNA ex-
tracted from dam?dcm?E. coli, requiring that DNA for
transformation be prepared from Dam methylation-deficient
strains of E. coli such as GM2163 (20). Therefore, it is prob-
able that B. anthracis encodes methylation-dependent restric-
tion enzymes (MDRs). We employed the Cre-lox method to
begin characterization of MDR genes as well as to obtain a
strain of B. anthracis more tractable to genetic manipulation.
Here, we report the sequential inactivation of the mrr and
mcrB genes, two of the three genes proposed in the online
restriction enzyme database (REBASE) as encoding MDRs.
This process not only resulted in the inactivation of the said
genes, but also led to excision of 30 kb of intervening genomic
sequence, thereby identifying 32 contiguous genes as being
dispensable for the growth of B. anthracis in rich media.
The methods developed here were also shown to be appli-
cable to modification of the large virulence plasmids of B.
anthracis. The four genes of the capBCAD region of the pXO2
plasmid are involved in synthesis and processing of the poly-
?-D-glutamic acid capsule. We introduced two loxP sites at the
boundaries of the region and used these to delete the entire
MATERIALS AND METHODS
Bacterial growth conditions and phenotypic characterization. E. coli strains
were grown in Luria-Bertani (LB) broth and used as hosts for cloning. LB agar
was used for selection of transformants (33). B. anthracis strains were grown in
brain heart infusion (BHI) and LB medium. Antibiotics (Sigma-Aldrich) were
added to media when appropriate to give the final concentrations indicated:
ampicillin (Ap; 100 ?g/ml, only for E. coli), erythromycin (Em; 400 ?g/ml for E.
coli, 5 ?g/ml for B. anthracis), spectinomycin (Sp; 100 ?g/ml for both E. coli and
B. anthracis). SOC medium (Quality Biologicals) was used for outgrowth of
transformation mixtures prior to plating on selective medium to isolate trans-
formants. B. anthracis colonies producing PA were identified by growth on CA
medium agar (36) supplemented with 0.8% (wt/vol) sodium bicarbonate, 5%
(vol/vol) horse serum, and 5% (vol/vol) sheep anti-PA serum (gift of J. Reimen-
schneider). On these plates, PA-producing colonies formed halo-like zones of
immunoprecipitation in as little as 12 h when grown in 20% CO2at 37°C.
Bicarbonate agar (10) with 0.8% (wt/vol) sodium bicarbonate and 10% (vol/vol)
equine serum was used for estimation of B. anthracis capsule production. Cap-
sule-producing strains formed mucoid colonies when grown in 20% CO2at 37°C
for 24 to 48 h. India ink was used to visually observe the presence of the B.
anthracis capsule (7). B. anthracis spores were prepared by growth on NBY-Mn
agar (nutrient broth, 8 g/liter; yeast extract, 3 g/liter; MnSO4· H2O, 25 mg/liter;
agar, 15 g/liter) at 30°C for 5 days (35). Congo red agar (39) was used for the
differentiation of B. anthracis Spo0A mutants (pink colonies). The capsules,
spores, and vegetative cells of B. anthracis were visualized on a Nikon Eclipse
E600W light microscope and photographed with a digital DXM1200F camera
(Nikon Instrument Inc., New York). Casein agar (1) was used for the differen-
tiation of protease-deficient strains of B. anthracis.
DNA isolation and manipulation. Preparation of plasmid DNA from E. coli,
transformation of E. coli, and recombinant DNA techniques were carried out by
using standard procedures (33). E. coli XL2-Blue and SCS110 competent cells
were purchased from Stratagene, and E. coli TOP10 competent cells were from
Invitrogen. Recombinant plasmid construction was carried out in E. coli XL2-
Blue or TOP10. Plasmid DNA from B. anthracis was isolated according to the
protocol for the purification of plasmid DNA from Bacillus subtilis (QIAGEN).
Chromosomal DNA from B. anthracis was isolated with a Wizard genomic
purification kit (Promega) in accordance with the protocol for isolation of
genomic DNA from gram-positive bacteria. B. anthracis was electroporated with
unmethylated plasmid DNA isolated from E. coli SCS110. Electroporation-
competent B. anthracis cells were prepared as previously described (25). Restric-
tion enzymes, T4 ligase, Klenow fragment, and alkaline phosphatase were pur-
chased from MBI Fermentas or New England Biolabs. Taq polymerase kits were
purchased from TaKaRa Shuzo or Invitrogen/Life Technologies. Ready-To-Go
PCR beads (Amersham Biosciences) were used for DNA rearrangement analy-
sis. For routine PCR analysis, a single colony was suspended in 200 ?l of
Tris-EDTA buffer (33), pH 8.0, heated to 95°C for 45 s, and then cooled to room
temperature. Cellular debris was removed by centrifugation at 15,000 ? g for 10
min. One microliter of the lysate contained sufficient template to support a PCR
with the PCR beads. The GeneRuler DNA ladder mix (MBI Fermentas) or the
1-kb Plus ladder (Invitrogen) was used for determination of DNA fragment
length. All constructs were verified by DNA sequencing and/or restriction en-
zyme digestion. All plasmids used in this study and their relevant characteristics
are listed in Table 1. Oligonucleotide primers are listed in Table 2.
Construction of targeting vectors. The general scheme for B. anthracis gene
knockout using the Cre/Lox system, presented in Fig. 1, employs in its first step
plasmids we have designated generically as pDC, for double-crossover plasmids.
These were derived from one of two temperature-sensitive (ts) plasmids, pE194
or pWVO1. The ts replicon from pE194 was extracted from pYJ335 (12) and
used to make pUE1, which has permissive and restrictive temperatures of 37°C
and 43.5°C, respectively. Plasmids pWVO1 and pVE6007 (18) were precursors to
the highly ts plasmid used here, pHY304 (30), which has permissive and restric-
tive temperatures of 30°C and 37°C, respectively. In most cases, the pDC plas-
mids had the gene of interest disrupted by the “?-sp element,” which consists of
the spectinomycin resistance (Spr) gene (aad9) of Enterococcus faecalis followed
by transcriptional stops. Previously, Saile and Koehler (32) successfully used the
?-sp element for gene disruption. Two different approaches were used for
generating a loxP-flanked ?-sp cassette for insertion into the gene of interest. In
the first approach, the BamHI fragment of pJRS312 containing the ?-sp cassette
was introduced into the BamHI site of plasmid pBS246 between its two directly
repeated loxP sequences. The resulting plasmid, p?L, was used as a template for
amplification of the loxP–?-sp–loxP cassette. The two primer pairs LoxSB/
LoxEB and LoxSN/LoxEN were used to generate loxP–?-sp–loxP fragments for
insertion within the spoAO and pepM genes, at BglII and BamHI sites (spoOA)
or at the NdeI site (pepM). This produced plasmids that were designated
pS?L408 and pS?L304 (Table 1). In the second approach, we PCR amplified
the ?-sp element using primers with loxP sites added to their 5? ends (primers
Tn5loxPaad9 For and Tn5loxPaad9 Rev) (Table 2). The loxP sites introduced by
either method worked satisfactorily.
A plasmid for expression of Cre recombinase was constructed using a PCR
fragment containing the P1 cre gene amplified from pBS185 (Gibco-BRL/Life
Technologies) with primers CreS and CreE. The fragment was inserted into the
corresponding NdeI and HindIII sites on pAE5 (27) to place expression of cre
under the control of the pagA promoter. The BglII-HindIII fragment of the
resulting pAEC5 plasmid was inserted between the BamHI and HindIII sites of
pHY304 to produce the Cre-expressing ts plasmid pCrePA (Fig. 1).
Bacterial strains and isolation of mutants. The strains used and their relevant
characteristics are listed in Table 3. B. anthracis mutants were constructed by the
replacement of coding sequences with the ? element conferring spectinomycin
resistance (Fig. 1). The targeting constructs were electroporated into B. anthracis
with selection for erythromycin resistance. Erythromycin-resistant colonies were
transferred to agar containing spectinomycin, incubated at the restrictive tem-
perature, and again transferred onto fresh agar containing spectinomycin. After
the third passage, at least 50 colonies were screened for spectinomycin resistance
and erythromycin sensitivity. In some cases, additional passages were required to
obtain this phenotype. Additionally, in the cases of spo0A and pepM mutants,
Congo red and casein agar, respectively, were used for an initial screening for
double-crossover events. Colonies in which a double-crossover recombination
event was suspected were validated by PCR analysis.
For elimination of the spectinomycin resistance, plasmid pCrePA was electro-
porated into the isolates with selection for erythromycin resistance at 30°C.
Erythromycin-resistant colonies were transferred to antibiotic-free agar and in-
cubated at 37°C to eliminate pCrePA. Colonies were then patched to three
separate agar plates containing erythromycin, spectinomycin, or no antibiotic.
The entire process was repeated when constructing mutants targeted in two or
more genes. The presence of a single loxP site within the targeted gene(s) of the
fully antibiotic-sensitive single or double mutants was confirmed by PCR and/or
PCR and sequence analysis of chromosomal modifications. Primers used for
PCR analysis are described in Table 2, and the locations of the corresponding
sequences are shown in the figures. Primers PAF and PAR correspond to se-
quences within the pagA gene. Primers pXO2-AT-f1/pXO2-AT-r1 (13) were
used to confirm the presence of the plasmid pXO2.
The sequence at the single loxP site remaining within spo0A was determined by
VOL. 74, 2006GENOME ENGINEERING IN B. ANTHRACIS 683
inserting the PCR fragments generated from strains MSLL35, MSLL34, and
MSLL33 (using primers Spo1 and Spo2) into pCR2.1-TOPO (Invitrogen) and
sequencing the resulting plasmid (National Institute of Dental and Craniofacial
Research core facility, National Institutes of Health, Bethesda, MD). Similarly,
the loxP within the pepM gene was amplified from strains MSLL35, MSLL34, and
MSLL33 with primers M4F and M4R and sequenced.
For analysis of the genomic deletion in the mcrB mrr double mutant (strain
McrB3P-Mrr-L?30), the region encompassing the entire mcrB-to-mrr region was
amplified by PCR in the parent strain using overlapping primer pairs (IG-1
through IG-8; sequences available from the authors on request). The 30-kb
deletion in the double mutant was verified with a PCR using primers McrB3P
For1 and Mrr 5? GenP, and the resulting PCR product was gel purified and
submitted for direct sequencing by the DNA sequencing and synthesis facility at
Iowa State University, Ames.
Genes selected for inactivation. A number of genes on the B.
anthracis chromosome or virulence plasmid pXO2 were tar-
geted for inactivation in this study. They are identified here
TABLE 1. Plasmids used in this study
Plasmid Relevant characteristic(s)a
Reference or source
Contains two directly repeated loxP sites flanking a multiple cloning site; Aprin E. coli
pUC18 carrying an ? element with spectinomycin resistance marker aad9 (?-sp) from
Enterococcus faecalis; Sprin E. coli and B. anthracis
2.3-kb BamHI fragment with ?-sp from pJRS312 inserted between two directly repeated
loxP of pBS246
Contains 2.3-kb BamHI fragment with ?-sp from pJRS312 flanked by both 3? and 5?
spo0A sequences; spo0A::?-sp; Aprin E. coli; Tcrin B. anthracis; Sprin both E. coli
and B. anthracis
pUTE408 with 2.4-kb Bg/II PCR fragment of p?L that contains the loxP-flanked ?-sp;
the fragment was amplified with LoxSB-LoxEB primers and inserted into the BamHI
site in place of existing ?-sp; spo0A:: loxP-?-sp-loxP
Hybrid of pE194 with pUC19 plasmid
PvuII-fragment of pYJ335 containing both pE194 and pUC19 replicons; Aprin E. coli;
Emrin B. anthracis
pUE1 containing PvuII fragment of pS?L408 with spo0A, loxP; and ?-sp sequences;
Contains Emrgene and strongly temperature-sensitive pWVO1 replicon for both E. coli
and gram-positive bacteria; Emrin both E. coli and B. anthracis
pHY304 with PvuII fragment of pS?L408 containing spo0A, loxP, and ?-sp sequences;
the fragment was inserted into the SmaI site of pHY304; spo0A::loxP-?-sp-loxP
Cloning vector for PCR products; AprKmrin E. coli
Cloning vector for PCR products; Aprin E. coli
2.5-kb PCR fragment (M4S-M4E primers) containing whole pepM gene of B. anthracis
cloned into pCR2.1-TOPO
TOPO-M4 with 2.6-kb NdeI PCR fragment of p?L that contains the loxP-flanked ?-sp;
the fragment was amplified with LoxSN-LoxEN primers and inserted into the NdeI
site of pepM, pepM::loxP-?-sp-loxP
pUE1 containing PvuII fragment of TOPO-M?L with pepM, loxP, and ?-sp sequences;
pUE1 containing pXO2 PCR fragment (CAPF-CAPE primers) disrupted by loxP-?-sp-
pUE1 containing pXO2 PCR fragment (DEPF-DEPE primers) disrupted by loxP-?-sp-
loxP cassette; capD::loxP-?-sp-loxP
Contains whole Cre recombinase gene
Hybrid of pUB110 with pBR322 plasmid; contains promoter of B. anthracis pagA gene;
Aprin E. coli, Kmrin B. anthracis
pAE5 with 1.05-kb NdeI-HindIII PCR fragment of pBS185 that contains the entire Cre
recombinase gene; the fragment was amplified with CreS-CreE primers and inserted
into corresponding sites of pAE5
pHY304 with BglII-HindIII fragment of pAEC5 containing entire Cre recombinase gene
under control of pagA promoter; the fragment was inserted into BamHI-HindIII of
Self-ligated pGEM-T easy with (i) mrr 5? sequences amplified by primers mrr 5F and
mrr 5R and cloned into the ApaI-SacII sites; (ii) mrr 3? sequences amplified by mrr
3F and mrr 3R and cloned into the PstI-SalI sites; and (iii) the NotI fragment from
p?L (containing the loxP-?-sp-loxP cassette) cloned into the NotI site
pHY304 with the SalI fragment from pMrr?L consisting of mrr::loxP-?-sp-loxP cloned
into the SalI site
pGEM-T easy with (i) mcrB3P sequences amplified by McrB3P For1 and McrB3P Rev,
cloned by the T-A method; (ii) loxP-?-sp-loxP amplified by Tn5loxPaad9For and
Tn5loxPaad9Rev and cloned into the BstZ17I site of mcrB giving
pHY304 with the NoI fragment from pMcrB3P?L consisting of mcrB::loxP-?-sp-loxP,
cloned into the NotI site
J. R. Scott
p?L This work
pS?L408 This work
TOPO-M?L This work
pM?L1 This work
pDS2 This work
pCrePA This work
pMrr?L This work
pHYMrr?L This work
pMcrB3P?L This work
pHYMcrB3P?L This work
aAbbreviations: Apr, ampicillin resistant; Emr, erythromycin resistant; Kmr, kanamycin resistant; Tcr, tetracycline resistant.
684 POMERANTSEV ET AL.INFECT. IMMUN.
TABLE 2. Primers used in this study
Primer Sequencea(5?-3?) (location)
Relevant propertyRestriction site
Primer pair to amplify loxP–?-sp–loxP BglII
Primer pair to amplify loxP–?-sp–loxP
Primer pair to amplify Cre
Outer primer for analysis of
modifications in spo0A from 5?
Inner primer for analysis of
modifications in spo0A from 5?
Inner primer for analysis of
modifications in spo0A from 3?
Inner primer for analysis of
modifications in spo0A from 3?
Outer primer for analysis of
modifications in spo0A from 3?
?-sp inner primer for analysis of
modifications in spo0A and pepM
Outer primer for analysis of
modifications in pepM from 5?;
used to amplify pepM gene from 5?
Outer primer for analysis of
modifications in pepM from 3?;
used to amplify pepM gene from 3?
Inner primer for analysis of
modifications in pepM from 5?
Inner primer for analysis of
modifications in pepM from 3?
Used to amplify pXO2 region
between promoters and capB gene
Used to amplify pXO2 region
between promoters and capB gene
Used to amplify pXO2 capD gene
Used to amplify pXO2 capD gene
Inner primer for analysis of pXO1
pagA from 5?
Inner primer for analysis of pXO1
pagA from 3?
5? primer for detection of pXO2
3? primer for detection of pXO2
Primer pair to amplify sequences on
the 5? side of mrr
PstIPrimer pair to amplify sequences on
the 3? side of mrr
Primer pair to amplify aad9 from
pUTE29 such that the PCR
product has a Tn5-binding
sequence (lowercase) and a loxP
sequence (bold italics) on each end
GAACTTTTCACCGAATAATGCCACTG Mrr5? GenPPrimer pair for analysis of McrB-mrr
CCGTCCCAATGATTAACTTTAATACPrimer to amplify mcrB sequences
together with McrB3P For1
Internal primer for the mcrB gene;
used for sequencing deletion
product in the double mutant
aRestriction enzyme recognition sites are underlined.
VOL. 74, 2006GENOME ENGINEERING IN B. ANTHRACIS 685
with the gene designations assigned by The Institute for
Genomic Research (Rockville, Maryland; http://www.tigr.org)
for the “Ames ancestor” strain chromosome (accession
NC_007323). The spo0A gene (GBAA4394 or BA4394) en-
codes an essential sporulation gene that acts at the first step of
sporulation. The pepM gene (GBAA0599) encodes a Zn2?
binding protease, also termed neutral peptidase/protease, and
is designated peptidase M04.012 in the MEROPS peptidase
database (http://merops.sanger.ac.uk). The mrr (GBAA2317)
and mcrB (GBAA2283) genes were initially suggested as being
involved in DNA restriction in the enzyme database main-
tained by New England Biolabs (http://rebase.neb.com). The
capBCAD region of plasmid pXO2 contains all four genes
(GBAA_pXO2_0063 to 0066) needed to synthesize and mod-
ify the extracellular poly-D-glutamic acid capsular material
Gene knockout using ts plasmids. Electroporation of B.
anthracis with plasmids in which the gene of interest was dis-
rupted with the ?-sp cassette led to efficient gene knockout, as
will be described with specific examples below. The strategy
used is shown in Fig. 1 and described in the Materials and
Methods section. Expression of the aad9 gene of the ?-sp
cassette was high enough to confer antibiotic resistance even
when present as a single copy integrated into the chromosome,
as demonstrated previously (32). The generic pDC targeting
plasmids we used contain the erm gene to allow selection for
the presence of the plasmid in B. anthracis. We found that 5
?g/ml of erythromycin is optimal for selection of transfor-
mants. An important attribute of the pDC plasmids is a tem-
perature-sensitive origin of replication. Previously, it has been
shown that two passages of B. anthracis cells containing pE194-
derivative plasmids at 43.5°C on LB agar without erythromycin
eliminated the plasmids (28). Here we successfully used the
pE194 replicon for ?-sp cassette insertion into B. anthracis.
However, we found that B. anthracis strain Ames 35 lost the
pXO1 plasmid when grown at 43.5°C, as was expected from
previous results (22). Therefore, we also used the more highly
ts replicon of plasmid pHY304 and found that a single passage
at 37°C entirely eliminated pHY304-derivative plasmids while
retaining pXO1. B. anthracis retained plasmid pXO2 during
passage at both 37 and 43.5°C. We designed the targeting
vectors to include loxP sites bracketing the ?-sp cassette so that
it could be removed by the site-specific Cre recombinase. Plas-
mid pCrePA was constructed to express the recombinase con-
stitutively in B. anthracis (Fig. 1) and to be easily removable by
growth at the restrictive temperature of the pHY304 replicon.
Insertional inactivation of the pepM and spo0A genes. We
disrupted the pepM and spo0A genes in three isogenic B. an-
thracis strains derived from the Ames strain (Table 3). We
chose these two genes because they have easily discernible
phenotypes, and also because the resulting mutated strains
have potential value as protein expression hosts. These genes
were interrupted individually and in combination by insertion
of the ?-sp cassette, as confirmed by PCR analysis (Fig. 2 and
3a and b). The PepM?phenotype was easily scored on plates
containing casein (see Fig. 5a, below) and the Spo0A?pheno-
type on plates containing Congo red agar (see Fig. 5b), as
previously described (39). The chromosomally integrated ?-sp
cassette was removed by introducing plasmid pCrePA, using
selection for erythromycin resistance at 30°C, and the isolated
colonies were then grown on antibiotic-free LB agar at 37°C.
We found that all of the colonies lost both the Emrand Spr
markers after overnight growth but retained the PepM?and/or
Spo0A?phenotypes. These results demonstrate that, in both
cases, the genes remained interrupted by the loxP site but the
Sprmarker was removed by the Cre function provided in trans
by pCrePA. Elimination of the Sprmarker was confirmed by
PCR analysis utilizing specific primers for each gene, as de-
picted in Fig. 2. Loss of pCrePA was phenotypically confirmed
by the inability of the clones to grow on LB plates containing
FIG. 1. Cre-loxP system for gene knockout in B. anthracis. (I) The
gene targeted for knockout is cloned into a ts plasmid and interrupted
by insertion of the loxP–?-sp–loxP (Spr) cassette. The plasmid is trans-
formed into B. anthracis, which is then grown at the restrictive tem-
perature. (II) The allelic exchange event is selected by the Sprpheno-
type, accompanied by loss of the pDC plasmid (and erythromycin
resistance). (III) Removal of the Sprcassette from the chromosome is
achieved by Cre-mediated recombination (excision) after transforming
the strain with pCrePA at 30°C. Cre recombinase expression plasmid
pCrePA contains the cre gene of bacteriophage P1 under control of the
B. anthracis protective antigen (pagA) gene promoter from pAE5 (27).
The signal peptide of protective antigen was eliminated in order to
retain Cre inside the cell. pCrePA also contains the Emrgene as a
selectable marker and the strongly ts replicon from pHY304 (30). (IV)
Growth at 37°C eliminates the Cre recombinase-producing ts vector
pCrePA. (V) The result is replacement of a portion of the targeted
gene by a single 34-bp loxP site.
686 POMERANTSEV ET AL.INFECT. IMMUN.
erythromycin. The absence of the vector in Emscells was also
confirmed by analysis of the plasmid content of the strains.
Only the original B. anthracis virulence plasmids were observed
in the B. anthracis double mutants (Fig. 3d).
Final corroboration of the chromosomal modifications in the
mutant strains was obtained by sequencing the chromosomal
DNA region containing the remaining loxP site. The nucleo-
tide sequences of these fragments demonstrated that, as ex-
pected, Cre catalyzed the perfect removal of the intervening
region, leaving only one copy of the loxP sequence. The result-
ing loxP sequence in the case of pepM disruption was an exact
match to the known sequence, whereas we found one change in
the case of spo0A (an A instead of G at the 27th position).
Further analysis of loxP sequences showed that this mutation
was generated in the right 13-bp RRinverted repeat of the
right loxP site during PCR amplification of the loxP–?-sp–loxP
fragment from p?L using the LoxSB/LoxEB primers. The mu-
tation was evidently retained during the Cre-mediated recom-
bination event. The scheme presented in Fig. 4 demonstrates
how a mutated recombinase binding site from the right loxPR
replaces a normal RLfrom the left loxPRin the single loxP site,
finally remaining within spo0A. Our data confirm that the right
inverted repeat of loxP is not as important for precise loxP
cleavage as the left one, consistent with prior evidence that Cre
initiates recombination of loxP by cleaving the upper strand on
the left loxP inverted repeat (21).
Phenotypes of pepM spo0A double mutants. The conse-
quences of disruption of both the pepM and spo0A genes were
the same in all three isogenic B. anthracis Ames derivative
strains. Mutation in pepM resulted in considerably decreased
proteolytic activities in comparison to the parent strain as
assessed on casein agar (Fig. 5a). Mutation in the spo0A gene
resulted in the inability of the mutant strain to form spores
(Fig. 5b). No colonies were obtained on either LB or BHI agar
TABLE 3. Strains used in this study
Strain Relevant characteristicsReference
B. anthracis strains
UM44-1C9 pXO1?pXO2?derivative of UM44-1; derived from Weybridge strain; requires indole;
pXO1?pXO2?Ames ANR-1 strain, produces PA
pXO1?pXO2?B. anthracis strain similar to ?Ames-1; produces capsule
pXO1?pXO2?Ames 34 derivative strain
Ames 35; spo0A knockout, Spr, containing two loxP sites; produces PA
Ames 34; spo0A knockout, Spr, containing two loxP sites; produces less capsule in comparison
with Ames 34
Ames 33; spo0A knockout, Spr, containing two loxP sites
S?L35; spo0A knockout containing one loxP site
S?L34; spo0A knockout containing one loxP site
S?L33; spo0A knockout containing one loxP site
Ames 35; pepM knockout, Spr, containing two loxP sites; produces PA
Ames 34; pepM knockout, Spr, containing two loxP sites; produces capsule similar with Ames 34
Ames 33; pepM knockout, Spr, containing two loxP sites
M?L35; pepM knockout containing one loxP site
M?L34; pepM knockout containing one loxP site
M?L33; pepM knockout containing one loxP site
ML35, Spr, spo0A knockout containing three loxP sites; produces PA
ML34, Spr, spo0A knockout containing three loxP sites; produces less capsule in comparison
with Ames 34
ML33, Spr, spo0A knockout containing three loxP sites
Ames 35; pepM knockout, spo0A knockout, residual loxP sites in both pepM and spo0A; retains
pXO1 plasmid; produces PA
Ames 34; pepM knockout, spo0A knockout, residual loxP sites in both pepM and spo0A; retains
pXO2 plasmid; produces less capsule in comparison with Ames 34
Ames 33; pepM knockout, spo0A knockout, residual loxP sites in both pepM and spo0A
Ames 34; Spr, containing two loxP sites; does not produce capsule
Ames 34; contains one loxP site; produces capsule
Ames 34; Spr, capD knockout, containing three loxP sites; produces less capsule in comparison
with Ames 34
Ames 34; contains one loxP site instead of the capBCAD region; does not produce capsule
pXO1?pXO2?Ind?Strrderivative of the Weybridge strain of B. anthracis
UM44-1C9; mcrB3P knockout, Sprflanked by two loxP sites
UM44-1C9; mcrB mrr double mutant, with a 30-kb deletion caused by Cre action, and a single
residual loxP site
MSLL34 This work
E. coli strains
TOP10F?mcrA ?(mrr-hsdRMS-mcrBC) ?80lacZ?M15 ?lacX74 deoR recA1 araD139 ?(ara-leu)7697
galU galK rpsL (StrR) endA1 nupG
recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F? proAB laclqZ?M15 Tn10 (Tcr) Amy Cmr]
rpsL (Smr) thr leu endA thi-1 lacY galK galT ara tonA tsx dam dcm supE44D (lac-proAB) [F?
traD36 proAB lacIqZDM15]
aStrain naming conventions: S denotes spoOA mutation, M denotes pepM mutation, L denotes the loxP site, and ? denotes spectinomycin insertion in the preceding
VOL. 74, 2006 GENOME ENGINEERING IN B. ANTHRACIS 687
after the MSLL33 strain was grown on NBY-Mn agar at 30°C
for 5 days and the bacteria were heated at 70°C for 30 min. An
aliquot of the same sample that was not heated at 70°C grew on
both LB and BHI agar. On the other hand, the parent Ames 33
strain formed heat-resistant spores at a high frequency when
subjected to identical treatment.
After PCR and phenotypic confirmation that the mutations
were as expected, we confirmed the retention of plasmids
pXO1 and pXO2 in the MSLL35 and MSLL34 mutants (Fig.
3d). Also, we analyzed PCR fragments amplified from pagA
located on pXO1 (38) and from the pXO2-at marker locus
(13). The sizes of the fragments were as expected (Fig. 3c).
However, we noted a reduced zone of PA immunoprecipita-
tion for the pXO1-containing double mutant and decreased
capsule production by the pXO2-containing double mutant
when these were compared to their respective parent strains.
Thus, MSLL34 exhibited capsule formation, but the India ink
particles were not fully excluded and the capsule material ap-
peared thinner than that on Ames 34 cells (Fig. 6a and b). We
suspect that these changes result from alterations in gene ex-
pression following the spo0A mutation. The affect of spo0A on
pagA gene expression may in part be due to abrB control by
spo0A (32). We also confirmed the stability of these mutations;
there was no alteration of the loxP site sequences in B. anthra-
cis MSLL33 following 30 passages at 37°C in either LB or BHI
Deletion of the capBCAD region of plasmid pXO2. The
structure of the capBCAD region as reported earlier (19) is
shown in Fig. 7. The capB, capC, capA, and capD genes are
transcribed in the same orientation under control of two tan-
dem promoters, P1 and P2, depending on CO2concentration.
Recent data on transcriptional analysis of the B. anthracis
capsule regulators demonstrated the presence of a third pro-
moter (P3) in the same area. It is interesting that transcription
from this promoter was not regulated by CO2(6).
Two plasmids, pCS1 and pDS2 (Table 1), were constructed
for introducing modification of the capBCAD region. The
pCS1 plasmid was used for insertion of the loxP–?-sp–loxP
cassette into the BglII site located between the P1/P2 promoter
site and the capB start codon. The pDS2 plasmid was used for
the replacement of a short BamHI fragment in the capD gene
by the same cassette (Fig. 7). Approximately 1 kb of homolo-
gous sequence was included on either side of the loxP–?-sp–
loxP cassette in the targeting vectors to ensure efficient double
crossover with either the capB or capD gene. Insertion of the
loxP–?-sp–loxP cassette into the BglII site located between the
P1/P2 promoter site and capB start codon (giving strain C?1)
prevented capBCAD operon expression even when the cells
FIG. 2. Knockout of pepM and spo0A genes. For each gene, the
diagrams show the native gene (a), the gene with an inserted ?-sp
cassette (b), and the gene after deletion of the ?-sp cassette (c).
Locations of the primers used for PCR analysis are shown by arrows.
Gray areas indicate the ?-sp cassette flanked by two loxP sites (b) and
residual loxP sequences resulting from the ?-sp cassette deletion (c).
FIG. 3. DNA analysis of modified B. anthracis strains. (a) pepM
gene amplified with M4S/M4E primers from Ames 33 (lane 1), strain
M?L33, containing the ?-sp cassette (lane 2), and strain MSLL33,
containing the deleted gene (lane 3). (b) spoOA gene amplified with
Spo1/Spo2 primers from Ames 33 (lane 1), strain S?L33, containing
the ?-sp cassette (lane 2), and strain MSLL33, containing the deleted
gene (lane 3). (c) pagA gene amplified with PAF/PAR primers from
Ames 35 (lane 1) and MSLL35 (lane 2) and pXO2 fragments amplified
with pXO2-AT-f1/pXO2-AT-r1 primers from Ames 34 (lane 3) and
MSLL34 (lane 4). (d) Virulence plasmid content of Ames 35 (lane 1),
MSLL35 (lane 2), Ames 34 (lane 3), and MSLL34 (lane 4). The arrows
indicate pXO1, pXO2, and chromosomal DNA bands, and the Mrlane
is a GeneRuler DNA ladder for comparison.
688 POMERANTSEV ET AL.INFECT. IMMUN.
were grown in 20% CO2. As a result of this insertion, B.
anthracis strain C?1 was unable to synthesize capsule (Fig. 7).
Cre-mediated excision of the ?-sp cassette from the C?1
pXO2 strain resulted in strain CL1, which possesses a pXO2
plasmid modified by insertion of a single loxP site between the
P1/P2 promoter site and the capB start codon. Although exci-
sion of the cassette from C?1 did restore capsule synthesis, the
resulting mutant, CL1, appears to produce less capsule than
the parent strain. This suggests that the palindromic loxP se-
quence (Fig. 4) might serve as a weak transcriptional termina-
tor. Subsequent insertion of the ?-sp cassette into the capD
gene in strain CL1 produced the strain designated CL1D?2,
which displayed an aberrant capsular morphology (Fig. 7). This
is likely due to the absence of Dep enzymatic activity, which
was reported to degrade high-molecular-weight capsule to a
lower-molecular-weight capsule (19). Other explanations for
the aberrant capsule on the mutant cells are suggested by the
work of Candela and Fouet, showing that CapD is required for
the covalent anchoring of capsule to peptidoglycan (5) and
work showing that CO2-mediated control of acpB occurs via
transcriptional read-through from atxA-dependent start sites of
capB (6). In those experiments, insertion of cassettes into the
capD gene was observed to either destabilize the capsule or
decrease transcription of capBCAD via acpB regulation. As a
result of loxP–?-sp–loxP insertion in the gene, strain CL1D?2
had three unidirectional loxP sites within the capBCAD region
(Fig. 7). Treatment with the Cre recombinase-expressing plas-
mid caused recombination between the outer LoxP sites and
deletion of the entire capBCAD region, leaving a single loxP
site. As expected, the resulting strain CL1DL2 had no ability to
form capsule (Fig. 7).
Construction of an mcrB-mrr double mutant strain. The
plasmid-free UM44-1C9 strain of B. anthracis was used in
experiments to identify genes involved in restriction of meth-
ylated DNA. From the published sequence for the Ames
strain, primers were designed to amplify mrr and mcrB se-
quences. Plasmid pHYMcrB3P?L (Table 1) was electropo-
rated into UM44-1C9, the resulting clones were propagated at
the restrictive temperature, and those with double-crossover
events were identified as detailed in the Materials and Meth-
ods section. An EmsSprclone was transformed with pCrePA
to eliminate the ?-sp cassette via Cre-mediated excision. This
was followed by curing the Spsclones of plasmid pCrePA by
means of propagation at the restrictive temperature, producing
strain McrB3P?L. Subsequently, strain McrB3P?L was trans-
formed with plasmid pHYMrr?L to disrupt the mrr gene by
double crossover, through insertion of the loxP–?-sp–loxP cas-
sette within the mrr gene. Expression of Cre recombinase in
FIG. 4. Schematic diagram of Cre-mediated excision of loxP–?-sp–loxP from the mutated B. anthracis spo0A gene. Cre recognizes and binds
to the 13-bp recombinase binding elements (RBEs) within the loxP site, which are arranged as inverted repeats surrounding a central 8-bp spacer
(shown in bold lowercase). The central 8 bp are asymmetric with respect to sequence and define the directionality of the site. Vertical arrows
indicate the cleavage sites for Cre-mediated recombination. The mutated base (A) in the right RBE of loxPRis shown in larger type and in boldface.
The corresponding, nonmutated base (G) in the right RBE of loxPLis shown in larger type. The mutated, right RBE from loxPRreplaced the
normal right RBE of loxPRin the single loxP site remaining in the spo0A gene as a result of Cre-mediated recombination, accompanied by the
excision of ?-sp, which contains the single loxP and cannot be replicated.
VOL. 74, 2006 GENOME ENGINEERING IN B. ANTHRACIS 689
this strain resulted in the excision of the ?-sp element, accom-
panied by deletion of the approximately 30 kb of chromosomal
DNA between the mcrB and mrr genes. The resulting strain
was designated McrB3P-Mrr-L?30. The analysis of the role of
the deleted genes in DNA restriction will be described else-
where (R. Sitaraman and S. H. Leppla, unpublished data).
Analysis of the 30-kb genomic deletion in strain McrB3P-
Mrr-L?30. To confirm that the mcrB-mrr region (nucleotides
2129380 to 2160611 in the “Ames ancestor” strain sequence;
accession number NC_007530) of the parent strain UM44-1C9
was similar to that of the Ames strain, PCR was done using
primer sets IG-1 to IG-8 designed to amplify overlapping sec-
tions of this region. These produced PCR products of the
expected sizes (Fig. 8a). Then, primers McrB3P For1 and Mrr
5? GenP were used to compare the intergenic region of strain
McrB3P-Mrr-L?30 to that of the parental strain, UM44-1C9.
The former strain yielded a PCR product close to the size
(2,074 bp) expected for the deletion (Fig. 8b). The parental
strain produced no product, as expected, because the condi-
tions used cannot amplify a 30-kb product. Direct sequencing
of the PCR product using primer McrB3P For2 further con-
firmed the presence of a single loxP site, flanked by remnants
of 5? sequences of the mrr and the mcrB genes, as well as a few
hundred bases of sequence derived either from the cloning
vectors used (pBS246 and pGEM-T easy) or from primer de-
sign (the Tn5-binding sequences). These data show that the
mcrB-mrr double mutant has a deletion spanning the entire
According to the annotations of the Ames genome provided
by The Institute for Genomic Research, the region between
mcrB and mrr contains 32 known and putative open reading
frames (ORFs), BA2284 to BA2316 (note: BA2285 is missing
in the database). In addition, in the deleted strain both the
mcrB and mrr genes (BA2283 and BA2317, respectively) are
truncated and, presumably, nonfunctional. We have therefore
identified a region of the B. anthracis chromosome spanning
31,231 bp and 34 ORFs which is dispensable for the growth of
B. anthracis in LB medium.
In the data presented here, genes in four different regions of
the B. anthracis chromosome and plasmids were inactivated by
homologous recombination. Thus, the Cre-loxP site-specific
FIG. 5. Phenotypic consequences of pepM and spo0A disruption in
B. anthracis. (a) Proteolysis of casein induced by B. anthracis MSLL33
(right side) is weak compared to the parent Ames 33 strain (left side).
For the test, 5 ?l of MSLL33 or Ames 33 overnight culture was spotted
on casein agar and grown for 12 h. (b) Congo red agar distinguishes the
parental B. anthracis strain (left side; Ames 33 strain) from the B.
anthracis spo0A mutant (right side; strain MSLL33). Ames 33 or
MSLL33 was streaked on the Congo red agar and grown for 24 h. Light
micrographs demonstrate either Ames 33 spores (left side) or remains
of nonsporulating MSLL33 vegetative cells (right side). Both strains
were grown at 30°C for 5 days on NBY-Mn agar.
FIG. 6. Comparison of PA and capsule production in parent and
mutated B. anthracis strains. (a) Immunoprecipitation of PA produced
by colonies of B. anthracis Ames 35 (pXO1?; left side) and the isogenic
double mutant MSLL35 (pXO1?; center), with Ames 34 (pXO1?;
right side) as a negative control. The cultures were grown for 18 h on
CA agar supplemented with 0.8% (wt/vol) sodium bicarbonate, 5%
(vol/vol) horse serum, and 5% (vol/vol) PA antiserum (from sheep) in
a 20% CO2environment at 37°C. (b) B. anthracis double mutant
MSLL34 (pXO2?) produces less capsule (right side) than the parent
Ames 34 strain (pXO2?; left side). The Ames 35 strain (pXO2?) was
used as a negative control (center). The cultures were grown for 18 h
in bicarbonate agar supplemented with 0.8% (wt/vol) bicarbonate and
10% (vol/vol) horse serum in 20% CO2at 37°C. Cells were removed
from the colonies shown, and capsule was visualized with India ink
(bottom panel). Bar, 5 ?m.
690 POMERANTSEV ET AL.INFECT. IMMUN.
recombination method used here provides a robust and easily
transferable approach to disrupting B. anthracis genes. The
availability of the complete genomic sequence of B. anthracis
(31) now makes it feasible to mutate any gene or putative ORF
to analyze its role, as has been extensively done for B. subtilis
(14). We have demonstrated the usefulness of the Cre-loxP
recombination system in B. anthracis by disrupting genes se-
quentially. These results establish that the system can yield
genetic modifications without the permanent establishment of
antibiotic resistance markers. Removal of the antibiotic resis-
tance markers assures that cell physiology will not be altered by
known or unrecognized activities of the resistance genes or the
antibiotic drugs themselves. Furthermore, the elimination of
antibiotic resistance markers may increase the acceptance of B.
anthracis for use in biotechnology applications, as in the pro-
duction of recombinant PA and lethal factor (16, 17).
This method could prove useful in making extensive modi-
fications to B. anthracis strains. For example, multiple pro-
teases could be inactivated so as to limit degradation of ex-
pressed proteins, as was done for B. subtilis (23). However, a
potential problem in this approach is that undesired recombi-
nation might occur between the loxP sites that accumulate
after multiple rounds of gene disruption. Recombination could
lead to deletion or inversion of large chromosomal segments,
depending on the orientation of the loxP sites, the distances
between them, and also the presence of essential genes in the
FIG. 7. Multistep deletion of the capBCAD region from the B. anthracis plasmid pXO2. The mutant strain C?1 was obtained as a result of
insertion of the loxP–?-sp–loxP cassette into the BglII site located between the P1/P2 promoter region and the capB start codon. Strain C?1 lost
the ability to synthesize capsule in contrast to the parent Ames 34 strain (micrographs on the right show India ink staining for capsule, as in Fig.
6). Cre-mediated excision of the ?-sp cassette from C?1 pXO2 resulted in strain CL1, containing a single loxP site (right-facing arrow) between
the promoters and the capB start codon. Capsule formation was restored in this strain. Insertion of the ?-sp cassette into the capD gene drastically
modified the capsule morphology of the resulting CL1D?2 strain. Cre treatment of CL1D?2 resulted in strain CL1DL2, which contains the pXO2
plasmid with a deleted capBCAD region. As a result of this deletion, the ability of strain CL1DL2 to synthesize capsule was completely lost.
VOL. 74, 2006GENOME ENGINEERING IN B. ANTHRACIS691
intervening region. To produce defined recombinational
events in strains containing multiple loxP sites, it may be pos-
sible to limit the activity of the Cre recombinase by growing the
pCrePA transformants at partially restrictive temperatures or
by expressing Cre from a tightly controlled, inducible pro-
In addition to use in inactivating individual genes, the intro-
duction of loxP sites offers a way of making large genome
rearrangements in the B. anthracis chromosome or virulence
plasmids, as was previously demonstrated in Lactococcus lactis
(3, 4). Because the efficiency of Cre-mediated recombination
does not depend on the distance between inverted loxP sites
(3), the Cre-loxP system offers a powerful tool for functional
analysis of large sets of B. anthracis genes. In the example
shown here, we used a single deletion event to show the non-
essentiality of 34 genes.
In the course of the work described here, we generated a
strain, B. anthracis MSLL33, which may prove useful as a host
for expression of recombinant proteins. The complete inability
of the strain to make spores prevents laboratory contamina-
tion. In our experience, B. anthracis spoOA mutant strains
grown in liquid culture die rapidly after reaching stationary
phase (data not shown). The inactivation of the major secreted
casein-degrading protease is likely to enhance the stability of
secreted proteins, thereby increasing their yield and integrity.
As noted above, the strain can be further improved by inacti-
vation of additional proteases.
The B. anthracis strain McrB3P-Mrr-L?30 may also have
potential value as a host strain for production of secreted
recombinant proteins. Although many of the 34 deleted genes
are annotated as “hypothetical,” several may alter relevant
phenotypes. BA2308 is predicted to encode the sporulation
control protein Spo0M. The B. anthracis Spo0M is highly ho-
mologous to the B. subtilis protein (62.3% similarity, 43.4%
identity) and is part of an operon consisting of genes BA2305
to BA2309. Upstream of BA2305 is the sequence 5?-AGGAT
ATGACCTATAAAAAAGAAAAACT-3?, in which the un-
derlined bases exactly match the consensus for ?H-dependent
promoters (29). B. subtilis spo0M mutants are susceptible to
lysis during growth and are impaired in their ability to sporu-
late (11). Another deleted gene that may affect sporulation is
BA2291, a KinA homolog (55.4% similarity, 34.6% identity
with the B. subtilis protein). KinA is a signal-transducing sensor
kinase that phosphorylates spo0F and thereby contributes to
the phosphorylation of Spo0A. B. subtilis kinA mutants are
delayed in the onset of sporulation (26). Two other deleted
genes (BA2288 and BA2310) encode proteins predicted to be
involved in regulating ion fluxes. Based on this information,
one could predict that strain McrB3P-Mrr-L?30 would be im-
paired in sporulation and osmotically fragile. Indeed, we found
visible clearing in saturated LB cultures of McrB3P-Mrr-L?30
left overnight at room temperature, as well as a reduction in
the number of viable cells compared to the parent strain,
UM44-1C9. Given the seemingly normal growth rate of
McrB3P-Mrr-L?30 in overnight cultures, its osmotic fragility,
and its impaired sporulation capabilities, this strain may be
termed a “crippled” avirulent strain and could therefore prove
suitable in situations where laboratory safety is a primary con-
cern. Another encouraging aspect is the transformability of
strain McrB3P-Mrr-L?30 with supercoiled, Dam-methylated
plasmid at reproducible, albeit low levels (R. Sitaraman, un-
published results). This strain can therefore be used as an
intermediate or final host for B. anthracis plasmids, especially
in situations wherein plasmid stability and replication fidelity
are of great importance.
This research was supported by the Intramural Research Program of
the NIH National Institute of Allergy and Infectious Diseases.
We thank Theresa Koehler (Department of Microbiology and Mo-
lecular Genetics, The University of Texas—Houston Health Science
Center, Houston) for providing plasmid pUTE408, Craig E. Rubens
(Department of Pediatrics, Division of Infectious Disease, Childrens’
Hospital and Regional Medical Center and University of Washington,
Seattle) for plasmid pHY304, and June R. Scott (Department of Mi-
crobiology and Immunology, Emory University School of Medicine,
Atlanta, Ga.) for plasmid pJRS312.
1. Aronson, A. I., N. Angelo, and S. C. Holt. 1971. Regulation of extracellular
protease production in Bacillus cereus T: characterization of mutants pro-
ducing altered amounts of protease. J. Bacteriol. 106:1016–1025.
2. Battisti, L., B. D. Green, and C. B. Thorne. 1985. Mating system for transfer
of plasmids among Bacillus anthracis, Bacillus cereus, and Bacillus thuringien-
sis. J. Bacteriol. 162:543–550.
3. Campo, N., M. L. Daveran-Mingot, K. Leenhouts, P. Ritzenthaler, and P. Le
Bourgeois. 2002. Cre-loxP recombination system for large genome rear-
rangements in Lactococcus lactis. Appl. Environ. Microbiol. 68:2359–2367.
4. Campo, N., M. J. Dias, M. L. Daveran-Mingot, P. Ritzenthaler, and P. Le
FIG. 8. Verification of 30-kb deletion in B. anthracis strain
McrB3P-Mrr-L?30. (a) Overlapping PCR products obtained from
DNA from the parent strain UM44-1C9 using primer pairs IG1 to IG8
(lanes 1 to 8, respectively). The mcrB-mrr intergenic segment is shown
as a shaded bar, and the mcrB and mrr genes are shown as antiparallel
shaded arrows. The thin lines represent the overlapping PCR products
obtained that collectively span the entire region. The loxP sites inserted
into the mcrB and mrr sequences to produce unmarked mutations are
shown as open arrows within the genes. Primers Mrr5? GenP and
McrB3P For1 (small antiparallel arrows above the mrr and mcrB
genes, respectively) produce no product from UM44-1C9 (lane 9). (b)
PCR with primers Mrr 5? GenP and McrB3P For1 results in a PCR
product only when McrB3P-Mrr-L?30 genomic DNA is used as a
template (lane 2), but not when UM44-1C9 genomic DNA or no DNA
is used as template (lanes 1 and 3, respectively). The corresponding
arrangement is shown in the schematic below the photograph. Mris the
1-kb Plus DNA ladder. The schematic diagrams are not drawn to scale.
692 POMERANTSEV ET AL.INFECT. IMMUN.
Bourgeois. 2004. Chromosomal constraints in gram-positive bacteria re- Download full-text
vealed by artificial inversions. Mol. Microbiol. 51:511–522.
5. Candela, T., and A. Fouet. 2005. Bacillus anthracis CapD, belonging to the
gamma-glutamyltranspeptidase family, is required for the covalent anchor-
ing of capsule to peptidoglycan. Mol. Microbiol. 57:717–726.
6. Drysdale, M., A. Bourgogne, and T. M. Koehler. 2005. Transcriptional anal-
ysis of the Bacillus anthracis capsule regulators. J. Bacteriol. 187:5108–5114.
7. Duguid, J. P. 1951. The demonstration of bacterial capsules and slime.
J. Pathol. Bacteriol. 63:673–685.
8. Farchaus, J. W., W. J. Ribot, S. Jendrek, and S. F. Little. 1998. Fermenta-
tion, purification, and characterization of protective antigen from a recom-
binant, avirulent strain of Bacillus anthracis. Appl. Environ. Microbiol. 64:
9. Fawcett, P., P. Eichenberger, R. Losick, and P. Youngman. 2000. The tran-
scriptional profile of early to middle sporulation in Bacillus subtilis. Proc.
Natl. Acad. Sci. USA 97:8063–8068.
10. Green, B. D., L. Battisti, T. M. Koehler, C. B. Thorne, and B. E. Ivins. 1985.
Demonstration of a capsule plasmid in Bacillus anthracis. Infect. Immun.
11. Han, W. D., S. Kawamoto, Y. Hosoya, M. Fujita, Y. Sadaie, K. Suzuki, Y.
Ohashi, F. Kawamura, and K. Ochi. 1998. A novel sporulation-control gene
(spo0M) of Bacillus subtilis with a sigmaH-regulated promoter. Gene 217:
12. Ji, Y., A. Marra, M. Rosenberg, and G. Woodnutt. 1999. Regulated antisense
RNA eliminates alpha-toxin virulence in Staphylococcus aureus infection. J.
13. Keim, P., L. B. Price, A. M. Klevytska, K. L. Smith, J. M. Schupp, R.
Okinaka, P. J. Jackson, and M. E. Hugh-Jones. 2000. Multiple-locus vari-
able-number tandem repeat analysis reveals genetic relationships within
Bacillus anthracis. J. Bacteriol. 182:2928–2936.
14. Kobayashi, K., S. D. Ehrlich, A. Albertini, G. Amati, K. K. Andersen, M.
Arnaud, K. Asai, S. Ashikaga, S. Aymerich, P. Bessieres, F. Boland, S. C.
Brignell, S. Bron, K. Bunai, J. Chapuis, L. C. Christiansen, A. Danchin, M.
Debarbouille, E. Dervyn, E. Deuerling, K. Devine, S. K. Devine, O. Dreesen,
J. Errington, S. Fillinger, S. J. Foster, Y. Fujita, A. Galizzi, R. Gardan, C.
Eschevins, T. Fukushima, K. Haga, C. R. Harwood, M. Hecker, D. Hosoya,
M. F. Hullo, H. Kakeshita, D. Karamata, Y. Kasahara, F. Kawamura, K.
Koga, P. Koski, R. Kuwana, D. Imamura, M. Ishimaru, S. Ishikawa, I. Ishio,
C. D. Le, A. Masson, C. Mauel, R. Meima, R. P. Mellado, A. Moir, S. Moriya,
E. Nagakawa, H. Nanamiya, S. Nakai, P. Nygaard, M. Ogura, T. Ohanan, M.
O’Reilly, M. O’Rourke, Z. Pragai, H. M. Pooley, G. Rapoport, J. P. Rawlins,
L. A. Rivas, C. Rivolta, A. Sadaie, Y. Sadaie, M. Sarvas, T. Sato, H. H.
Saxild, E. Scanlan, W. Schumann, J. F. Seegers, J. Sekiguchi, A. Sekowska,
S. J. Seror, M. Simon, P. Stragier, R. Studer, H. Takamatsu, T. Tanaka, M.
Takeuchi, H. B. Thomaides, V. Vagner, J. M. van Dijl, K. Watabe, A. Wipat,
H. Yamamoto, M. Yamamoto, Y. Yamamoto, K. Yamane, K. Yata, K. Yo-
shida, H. Yoshikawa, U. Zuber, and N. Ogasawara. 2003. Essential Bacillus
subtilis genes. Proc. Natl. Acad. Sci. USA 100:4678–4683.
15. Koehler, T. M., Z. Dai, and M. Kaufman-Yarbray. 1994. Regulation of the
Bacillus anthracis protective antigen gene: CO2and a trans-acting element
activate transcription from one of two promoters. J. Bacteriol. 176:586–595.
16. Leppla, S. H. 2000. Anthrax toxin, p. 445–472. In K. Aktories and I. Just
(ed.), Bacterial protein toxins. Springer, Berlin, Germany.
17. Leppla, S. H., J. B. Robbins, R. Schneerson, and J. Shiloach. 2002. Devel-
opment of an improved vaccine for anthrax. J. Clin. Investig. 110:141–144.
18. Maguin, E., P. Duwat, T. Hege, D. Ehrlich, and A. Gruss. 1992. New ther-
mosensitive plasmid for gram-positive bacteria. J. Bacteriol. 174:5633–5638.
19. Makino, S., M. Watarai, H. I. Cheun, T. Shirahata, and I. Uchida. 2002.
Effect of the lower molecular capsule released from the cell surface of
Bacillus anthracis on the pathogenesis of anthrax. J. Infect. Dis. 186:227–233.
20. Marrero, R., and S. L. Welkos. 1995. The transformation frequency of
plasmids into Bacillus anthracis is affected by adenine methylation. Gene
21. Martin, S. S., E. Pulido, V. C. Chu, T. S. Lechner, and E. P. Baldwin. 2002.
The order of strand exchanges in Cre-LoxP recombination and its basis
suggested by the crystal structure of a Cre-LoxP Holliday junction complex.
J. Mol. Biol. 319:107–127.
22. 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:
23. Murashima, K., C. L. Chen, A. Kosugi, Y. Tamaru, R. H. Doi, and S. L.
Wong. 2002. Heterologous production of Clostridium cellulovorans engB,
using protease-deficient Bacillus subtilis, and preparation of active recombi-
nant cellulosomes. J. Bacteriol. 184:76–81.
24. Palmeros, B., J. Wild, W. Szybalski, S. Le Borgne, G. Hernandez-Chavez, G.
Gosset, F. Valle, and F. Bolivar. 2000. A family of removable cassettes
designed to obtain antibiotic-resistance-free genomic modifications of Esch-
erichia coli and other bacteria. Gene 247:255–264.
25. Park, S., and S. H. Leppla. 2000. Optimized production and purification of
Bacillus anthracis lethal factor. Protein Expr. Purif. 18:293–302.
26. Perego, M., S. P. Cole, D. Burbulys, K. Trach, and J. A. Hoch. 1989. Char-
acterization of the gene for a protein kinase which phosphorylates the sporu-
lation-regulatory proteins Spo0A and Spo0F of Bacillus subtilis. J. Bacteriol.
27. Pomerantsev, A. P., K. V. Kalnin, M. Osorio, and S. H. Leppla. 2003.
Phosphatidylcholine-specific phospholipase C and sphingomyelinase activi-
ties in bacteria of the Bacillus cereus group. Infect. Immun. 71:6591–6606.
28. Pomerantsev, A. P., and N. A. Staritsyn. 1996. Povedenie geterologichnoi
rekombinantnoi plasmidy pCET v kletkakh Bacillus anthracis. Genetika 32:
29. Predich, M., G. Nair, and I. Smith. 1992. Bacillus subtilis early sporulation
genes kinA, spo0F, and spo0A are transcribed by the RNA polymerase
containing sigma H. J. Bacteriol. 174:2771–2778.
30. Pritzlaff, C. A., J. C. Chang, S. P. Kuo, G. S. Tamura, C. E. Rubens, and V.
Nizet. 2001. Genetic basis for the beta-haemolytic/cytolytic activity of group
B Streptococcus. Mol. Microbiol. 39:236–247.
31. Read, T. D., S. N. Peterson, N. Tourasse, L. W. Baillie, I. T. Paulsen, K. E.
Nelson, H. Tettelin, D. E. Fouts, J. A. Eisen, S. R. Gill, E. K. Holtzapple,
O. A. Okstad, E. Helgason, J. Rilstone, M. Wu, J. F. Kolonay, M. J. Beanan,
R. J. Dodson, L. M. Brinkac, M. Gwinn, R. T. DeBoy, R. Madpu, S. C.
Daugherty, A. S. Durkin, D. H. Haft, W. C. Nelson, J. D. Peterson, M. Pop,
H. M. Khouri, D. Radune, J. L. Benton, Y. Mahamoud, L. Jiang, I. R. Hance,
J. F. Weidman, K. J. Berry, R. D. Plaut, A. M. Wolf, K. L. Watkins, W. C.
Nierman, A. Hazen, R. Cline, C. Redmond, J. E. Thwaite, O. White, S. L.
Salzberg, B. Thomason, A. M. Friedlander, T. M. Koehler, P. C. Hanna,
A. B. Kolsto, and C. M. Fraser. 2003. The genome sequence of Bacillus
anthracis Ames and comparison to closely related bacteria. Nature 423:81–
32. Saile, E., and T. M. Koehler. 2002. Control of anthrax toxin gene expression
by the transition state regulator abrB. J. Bacteriol. 184:370–380.
33. Sambrook, J. and D. W. Russell. 2001. Molecular cloning: a laboratory
manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
34. Sternberg, N. 1981. Bacteriophage P1 site-specific recombination. III. Strand
exchange during recombination at lox sites. J. Mol. Biol. 150:603–608.
35. Thorne, C. B. 1993. Bacillus anthracis, p. 113–124. In A. B. Sonenshein, J. A.
Hoch, and R. Losick (ed.), Bacillus subtilis and other gram-positive bacteria:
biochemistry, physiology, and molecular genetics. American Society for Mi-
crobiology, Washington, D.C.
36. Thorne, C. B., and F. C. Belton. 1957. An agar-diffusion method for titrating
Bacillus anthracis immunizing antigen and its application to a study of anti-
gen production. J. Gen. Microbiol. 17:505–516.
37. Thwaite, J. E., L. W. Baillie, N. M. Carter, K. Stephenson, M. Rees, C. R.
Harwood, and P. T. Emmerson. 2002. Optimization of the cell wall micro-
environment allows increased production of recombinant Bacillus anthracis
protective antigen from B. subtilis. Appl. Environ. Microbiol. 68:227–234.
38. Vodkin, M. H. and S. H. Leppla. 1983. Cloning of the protective antigen gene
of Bacillus anthracis. Cell 34:693–697.
39. Worsham, P. L., and M. R. Sowers. 1999. Isolation of an asporogenic
(spoOA) protective antigen-producing strain of Bacillus anthracis. Can. J.
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