Single-copy chromosomal integration systems for
Eric D. LoVullo,1Claudia R. Molins-Schneekloth,23 Herbert P. Schweizer2
and Martin S. Pavelka, Jr1
Martin S. Pavelka, Jr
1Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester,
NY 14642, USA
2Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins,
CO 80523, USA
Received 21 July 2008
Revised8 December 2008
Accepted 29 December 2008
Francisella tularensis is a fastidious Gram-negative bacterium responsible for the zoonotic
disease tularemia. Investigation of the biology and molecular pathogenesis of F. tularensis has
been limited by the difficulties in manipulating such a highly pathogenic organism and by a lack of
genetic tools. However, recent advances have substantially improved the ability of researchers to
genetically manipulate this organism. To expand the molecular toolbox we have developed two
systems to stably integrate genetic elements in single-copy into the F. tularensis genome. The first
system is based upon the ability of transposon Tn7 to insert in both a site- and orientation-specific
manner at high frequency into the attTn7 site located downstream of the highly conserved glmS
gene. The second system consists of a sacB-based suicide plasmid used for allelic exchange of
unmarked elements with the blaB gene, encoding a b-lactamase, resulting in the replacement of
blaB with the element and the loss of ampicillin resistance. To test these new tools we used them
to complement a novel D-glutamate auxotroph of F. tularensis LVS, created using an improved
sacB-based allelic exchange plasmid. These new systems will be helpful for the genetic
manipulation of F. tularensis in studies of tularemia biology, especially where the use of multi-copy
plasmids or antibiotic markers may not be suitable.
Tularemia has a variety of clinical manifestations depend-
ing on route of entry, subspecies of bacteria and inoculum
size, while the disease state can range from mild to fatal
(Ellis et al., 2002). There are three official subspecies of the
causative agent Francisella tularensis: the highly pathogenic
F. tularensis subsp. tularensis, and the less pathogenic F.
tularensis subsp. holarctica and F. tularensis subsp.
mediaasiatica. The high mortality of F. tularensis subsp.
tularensis pneumonic tularemia and its high infectivity has
raised concerns about its potential use as a biological
weapon (Dennis et al., 2001). These concerns have
prompted the US Centers for Disease Control (CDC) to
classify F. tularensis as a Select Agent.
A wide variety of genetic tools have recently been
developed for the manipulation of Francisella. These
include Escherichia coli–Francisella shuttle vectors (Bina
et al., 2006; LoVullo et al., 2006; Ludu et al., 2008; Maier
et al., 2004; Rasko et al., 2007), random transposon
mutagenesis systems based on EZ-Tn5, Himar1 and Tn5
(Buchan et al., 2008; Kawula et al., 2004; LoVullo et al.,
2006; Maier et al., 2006; Qin & Mann, 2006), as well as
methods for allelic exchange (Golovliov et al., 2003;
LoVullo et al., 2006; Ludu et al., 2008; Rodriguez et al.,
2008; Twine et al., 2005). One methodology that has not
been fully explored is the integration of genetic elements
into the F. tularensis chromosome. In other bacteria this
has been accomplished using non-replicative vectors
containing an attachment site and integrase gene from a
lysogenic bacteriophage (Hoang et al., 2000; Stover et al.,
1991). This approach is not possible for F. tularensis, since
phages that are infective for this organism have yet to be
In this paper, we report the development of two single-
copy integration systems for incorporating genetic ele-
ments into the Francisella genome. The first system is based
on the transposon Tn7 and takes advantage of its ability to
insert in both a site- and orientation-specific manner at
high frequency into the attTn7 site, located downstream of
the highly conserved glmS gene, which encodes the
essential glucosamine-6-phosphate synthetase (Peters &
Craig, 2001). This system has been used in a number of
Abbreviations: D-AAT, D-amino acid transferase; PGA, poly-c-D-glutamic
3Present Address: Centers for Disease Control and Prevention, Division
of Vector-Borne Infectious Diseases, Bacterial Diseases Branch, Fort
Collins, CO 80521, USA.
Microbiology (2009), 155, 1152–1163
1152 022491G2009 SGM Printed in Great Britain
pathogens, including Pseudomonas aeruginosa, E. coli,
Burkholderia mallei, Burkholderia
Yersinia pestis (Choi et al., 2005, 2006, 2008; McKenzie &
Craig, 2006). As the insertion occurs in an intergenic
region the fitness of the modified organisms appears to be
unchanged (Choi et al., 2005; Peters & Craig, 2001).
We constructed a mini-Tn7 vector, which has a kanamy-
cin-resistance marker flanked by cd-res sites, and a helper
plasmid, which encodes the site-specific Tn7 transposase
complex TnsABCD, expressed from an F. tularensis
promoter. We confirmed the ability of the mini-Tn7 to
stably insert at the attTn7 site in the F. tularensis
chromosome. We also showed that the kanamycin marker
can be efficiently excised by the cd-resolvase. The ability to
remove the kanamycin marker is important, because F.
tularensis genetics has a limited repertoire of Select Agent
approved markers (Titball et al., 2007).
The second system uses a sacB-based suicide plasmid
expressing kanamycin resistance that is used for allelic
exchange of unmarked elements with the blaB gene, which
encodes the only functional b-lactamase in F. tularensis
(Bina et al., 2006; LoVullo et al., 2006). The deletion of the
blaB gene allows for convenient screening of desired
recombinants based on their sensitivity to ampicillin.
Bacterial strains, culture conditions, and transformation. E. coli
DH10B (Table 1) was used for routine cloning procedures and was
grown in Luria–Bertani (LB) broth (BD Biosciences) or on LB agar. E.
coli HB101 was used to maintain all plasmids containing the cd-res
cassettes and was grown as described above. F. tularensis strains
(Table 1) were grown as previously reported (LoVullo et al., 2006).
Specifically, strains were grown at 37 uC in liquid modified Mueller–
Hinton medium (MMH), which is Mueller–Hinton broth (BD
Biosciences) supplemented with 1.0% (w/v) glucose, 0.025% (w/v)
ferric pyrophosphate (Sigma-Aldrich) and 0.05% (w/v) L-cysteine
free base (Calbiochem), or on MMH agar, which is the MMH
medium described above supplemented with 1.0% (w/v) proteose
peptone (BD Biosciences), 2.5% (v/v) defibrinated sheep blood
(Remel) and 1.5% (w/v) bacto-agar (BD Biosciences). When
necessary, ampicillin (Ap; Sigma-Aldrich) was added at 100 or
50 mg ml21, respectively, for E. coli or F. tularensis, while kanamycin
(Km; Sigma-Aldrich) was used at 50 mg ml21for E. coli and 5 mg
ml21for F. tularensis strains LVS and Schu. Kanamycin stock
solutions were made by accounting for the concentration of active
kanamycin in each lot. Hygromycin B (Hyg; Roche Applied Science)
was used at 200 mg ml21for all species and strains. Sucrose was used
at a final concentration of 8 or 5% (w/v) depending on the sacB
vector. The b-galactosidase substrate X-Gal (Invitrogen) was used at
50 mg ml21in MMH agar lacking sheep blood. D-Glutamic acid
(Sigma-Aldrich) was used at a final concentration of 200 mg ml21in
MMH broth and in MMH agar lacking proteose peptone and sheep
Electroporations and allelic exchange experiments were done as
described previously (LoVullo et al., 2006).
DNA manipulation. DNA methods were performed essentially as
described by Ausubel et al. (1987). DNA fragments were isolated
using agarose gel electrophoresis and QIAquick spin columns
(Qiagen). Oligonucleotides were synthesized by Invitrogen Life
Technologies. Oligonucleotides flanking the Tn7 attachment site
were attF, 59-ATGCAGGACATGATTTTAGTG
attTn7), and attR, 59-TTATGTTGAGTCCATATTTCAG (reverse 39
to attTn7). All restriction endonucleases and DNA modifying or
polymerase enzymes were from New England Biolabs or Fermentas.
PCRs were performed with Iproof High-Fidelity DNA Polymerase
(Bio-Rad) according to the manufacturer’s recommendations. All
plasmids used in this study (Table 1) were from the authors’
collections. Preparation of plasmid and genomic DNA from E. coli
and F. tularensis was done as previously reported (LoVullo et al.,
(forward 59 to
b-Galactosidase assay. The assays were performed on whole cell
suspensions according to a standard protocol (Miller, 1972).
Plasmid construction. Plasmids used in this study are described in
Table 1. Detailed descriptions of the construction of the plasmids
used in this study can be obtained from the corresponding author.
Information about plasmid construction that is pertinent to the
understanding of this work is described below.
Unstable replicating helper plasmid, pMP720 (Fig. 1a). The R6K
origin from plasmid pTNS2 (accession no. AY884833) was replaced
with the pUC ori from pBluescript II KS(+) to produce pMP650. The
tnsABCD operon from pMP650 was cloned into the multiple cloning
site of pMP658 to produce pMP685. Inverse PCR was performed on
pMP685, eliminating 380 bp between the tnsABCD operon and the
blaB promoter, producing pMP720.
Non-replicative mini-Tn7 vector, pMP749 (Fig. 1b). The R6K
origin from plasmid pUC18R6KT mini-Tn7T (GenBank accession no.
AY712953) was replaced with the pUC ori from pBluescript II KS(+)
to produce the empty mini-Tn7 pMP651. The PgroESL–aphA-1 from
pMP527 was flanked with cd-res sites and ligated into pMP651 to
Non-replicative mini-Tn7 GFP vector, pMP793 (Fig. 1c). The R6K
origin from plasmid pUC18R6KT mini-Tn7T gnELp-Kan-GFP was
replaced with the pUC ori from pBluescript II KS(+) to produce
pMP661. The F. tularensis rpsL promoter region was amplified from
Schu genomic DNA by PCR and placed upstream of gfp, encoding
GFPmut3 (Cormack et al., 1996), which produced pMP761. This
PrpsL–gfp fragment was then inserted into the multiple cloning site of
pMP749, generating pMP793.
Unstable cd-resolvase plasmid, pMP672 (Fig. 1d). The cd-
resolvase gene, tnpR, was obtained from pGH542 and inserted into
the multiple cloning site of pMP658 to generate pMP672.
blaB integration vectors, pMP719, pMP790 and pMP815 (Fig. 4).
Inverse PCR was performed on the suicide vector pMP590 to
eliminate the pFNL10 ori yielding pMP671. The flanking regions of
blaB were amplified from Schu genomic DNA and cloned
upstream (59 flanking) and downstream (39 flanking) of the
multiple cloning site of suicide vector pMP671 to produce the blaB
integration vector pMP719. The lacZ gene was cloned into the
multiple cloning site of pMP719 to form pMP741, and then the
rpsL promoter region was amplified from Schu genomic DNA and
placed upstream of the lacZ gene to form pMP790. This construct
was created to test for heterologous gene expression in the blaB
To improve the counterselectable marker, the repA promoter region
upstream of sacB of pMP719 was removed by restriction digestion.
This region was replaced with the dnaK promoter region amplified
F. tularensis integration systems
from F. tularensis Schu genomic DNA using PCR and cloned
upstream of sacB to form pMP799. Inverse PCR was then performed
with pMP799 to eliminate 192 bp of unnecessary DNA upstream of
PdnaKto produce pMP815.
Modified sacB suicide vector, pMP812 (Fig. 6a). The Francisella
ori region and repA promoter region upstream of sacB were removed
from suicide vector pMP590 by restriction digestion. This region was
replaced with the dnaK promoter and cloned upstream of sacB to
Table 1. Bacterial strains and plasmids
Sources: J. Benach, State University of New York (SUNY), Stony Brook, NY, USA; M. Schriefer, CDC, Fort Collins, CO, USA.
Strain or plasmidDescription Reference or source
E. coli strains
DH10BF2mcrA D(mcrBC-hsdRMS-mrr) [w80dDlacZDM15] DlacX74 deoR recA1 endA1
araD139 D(ara,leu)7697 galU galK l2rpsL nupG
F2D(gpt-proA)62 leuB1 glnV44 ara-14 galK2 lacY1 hsdS20 rpsL20 xyl-5 mtl-1
Boyer & Roulland-
F. tularensis strains
pBluescript II KS (+)
F. tularensis subsp. holarctica live vaccine strain
F. tularensis subsp. tularensis
LVS DmurI1 attTn7::PrpsL–murI+res-aphA-1-res
LVS DmurI1 attTn7::PrpsL–murI+res
LVS DmurI1 DblaB::PrpsL–murI+
ApR, cloning vector
TcR, source of tnpR
ApR, R6K replicon, mini-Tn7 base vector
ApR, KmR, R6K replicon, mini-Tn7 gfp
Piuri & Hatfull (2006)
Choi et al. (2005)
ApR, R6K replicon, plasmid expressing tnsABCD from Plac
KmR, E. coli–F. tularensis shuttle vector with the PgroESL–aphA-1 cassette
HygR, E. coli–F. tularensis shuttle vector with the PgroESL–hyg cassette
KmR, F. tularensis sacB suicide vector
ApR, plasmid expressing tnsABCD from Plac
ApR, mini-Tn7 base vector
HygR, pMP529 with PblaBupstream of a multiple cloning site
ApR, KmR, mini-Tn7 gfp
KmR, sacB suicide vector lacking pFLN10 ori derived from pMP590
HygR, pMP658 with PblaB–tnpR
HygR, pMP658 with PblaB–tnsABCD
KmR, sacB-based blaB integration vector
HygR, pMP685 with PblaBmoved 380 bp closer to tnsABCD
KmR, sacB-based blaB integration vector based on pMP719 with lacZ in the
multiple cloning site
ApR, KmR, mini-Tn7 vector with res-aphA-1-res
ApR, KmR, mini-Tn7 with PrpsL–gfp
KmR, E. coli–F. tularensis shuttle vector with PrpsL–gfp
KmR, sacB suicide vector derived from pMP590 with PdnaK–sacB
KmR, blaB integration vector based on pMP741 with PrpsL–lacZ
ApR, KmR, pMP749 with PrpsL–gfp in the multiple cloning site
KmR, blaB integration vector derived from pMP719 with PdnaK–sacB
KmR, 192 bp smaller, improved sacB suicide vector derived from pMP780
KmR, 192 bp smaller, improved blaB integration vector derived from pMP719
KmR, pMP812 suicide vector bearing the murI region of LVS
KmR, pMP812 suicide vector containing DmurI allele derived from pMP880
ApR, KmR, pMP749 with PrpsL–murI+from pMP889 in the multiple cloning site
KmR, pMP815 with PrpsL–murI+from pMP889 in the multiple cloning site
Choi et al. (2005)
LoVullo et al. (2006)
LoVullo et al. (2006)
LoVullo et al. (2006)
LoVullo et al. (2008)
E. D. LoVullo and others
1154 Microbiology 155
Fig. 1. Maps of the Tn7 system vectors. (a) The helper plasmid pMP720 is an unstable E. coli–F. tularensis shuttle vector that contains the Francisella blaB promoter driving
the site- and orientation-specific transposase complex tnsABCD for integrating (b) the mini-Tn7 pMP749, which contains the transposon end Tn7L, a multiple cloning site,
two terminators (T1and T0) to limit readthrough from the glmS promoter on insertion, PgroESL–aphA-1 (conferring resistance to kanamycin flanked by the cd-sites for resolution
of the cassette) and the other transposon end Tn7R, or (c) pMP793, which has the Francisella rpsL promoter driving gfp expression in the multiple cloning site of pMP749. (d)
The resolvase plasmid pMP672 is an unstable E. coli–F. tularensis shuttle vector which has tnpR, encoding cd-resolvase, driven by the blaB promoter for resolution of the
kanamycin cassette after insertion into the Francisella genome.
F. tularensis integration systems
form pMP780. Inverse PCR was performed as above to eliminate
192 bp of unnecessary DNA upstream of PdnaKto produce pMP812.
Plasmid for DmurI allelic exchange, pMP884. A DNA fragment
containing murI was obtained from strain LVS genomic DNA using
PCR and cloned into pMP812 to yield pMP880. An in-frame deletion
of 768 bp within murI was made using PCR to yield pMP884. There
are 962 bp upstream and 982 bp downstream of the DmurI1 allele in
Non-replicative mini-Tn7 murI complementing vector, pMP890.
The F. tularensis murI gene was amplified from LVS genomic DNA by
PCR and placed downstream of PrpsLin pMP767, forming pMP889.
This PrpsL–murI fragment was then inserted into the multiple cloning
site of pMP749, generating pMP890.
blaB::murI+integration vector, pMP895. The PrpsL–murI frag-
ment was amplified from pMP889 by PCR and inserted into the
multiple cloning site of pMP815, generating pMP895.
RESULTS AND DISCUSSION
Development of the mini-Tn7 system
The most common implementation of the Tn7 system is to
simultaneously deliver both transposon and transposase to
the cells on separate suicide plasmids (Choi et al., 2005).
This allows for transient expression of the transposase and
subsequent integration of the transposon in the chro-
mosome without replication of the delivery plasmids. This
approach did not work with F. tularensis using the suicide
transposase plasmid pTNS2 (Table 1) and an early
generation Tn7 suicide plasmid. We first hypothesized
that the lac promoter driving the 6 kb tnsABCD operon in
the helper plasmid was not functional in F. tularensis. We
created another suicide helper plasmid with the operon
cloned downstream of the Francisella groESL promoter,
which is the same promoter that we have used for the
expression of selectable markers. However, this approach
was also unsuccessful. We found that the tnsABCD genes
are not optimal for the codon preference of F. tularensis
and this, coupled with the size of the operon, probably
prevented the cells from producing enough transposase
proteins to catalyse transposition during the transient
expression period. We then hypothesized that expressing
the operon from a replicating plasmid prior to introduc-
tion of the Tn7 suicide plasmid would allow time for the
cell to produce sufficient amounts of transposase. A similar
method using a temperature-sensitive helper plasmid has
been reported to express the transposase in E. coli and S.
typhimurium (McKenzie & Craig, 2006). Toward this end,
we created the helper plasmid pMP720 (Fig. 1a) from the
unstable hygromycin-resistant shuttle plasmid pMP658
(LoVullo et al., 2008), which contains the transposase
operon expressed from the Francisella blaB promoter. This
revised strategy proved successful, as described below.
In addition to the helper plasmid, we created a mini-Tn7
element on a suicide vector. The plasmid pMP749 (Fig. 1b)
contains the kanamycin-resistance marker aphA-1 driven
by the Francisella groESL promoter, flanked by cd-res DNA
binding sites for the site-specific cd-resolvase of E. coli
transposon Tn1000 (Bardarov et al., 2002). It also contains
two terminators (T0and T1) to prevent read-through from
the glmS promoter after chromosomal insertion (Choi et
al., 2005) and a multiple cloning site for cloning DNA
elements. An additional mini-Tn7 construct, pMP793 (Fig.
1c), was made to express GFP, and has a Francisella rpsL
promoter driving gfp cloned into the multiple cloning site
The methodology we developed is shown in Fig. 2. First,
the helper plasmid pMP720 is electroporated into F.
tularensis and transformants selected by hygromycin
resistance. One clone is prepared for electroporation while
maintaining hygromycin selection. We then transform the
strain with the mini-Tn7 plasmid pMP749, and select for
kanamycin-resistant clones. We routinely obtain ~104
kanamycin-resistant LVS transformants per electropora-
tion with ~1 mg pMP749 DNA. We obtained similar results
with the mini-Tn7 plasmid expressing GFP, pMP793, with
both LVS and Schu, resulting in ~104kanamycin-resistant
transformants per electroporation. We grew kanamycin-
resistant transformants overnight in liquid media lacking
selection and these were subcultured 1:10 and grown for
an additional 24 h, after which they were plated on
medium lacking antibiotics. The antibiotic-resistance
phenotypes of the resulting clones were then screened,
and we found that the hygromycin-resistance helper
plasmid pMP720 was lost from the population at a
encoded in Tn7, was maintained at 100% in the
population. This confirmed our expectations that the
helper plasmid would be readily lost from the population
but that the Tn7 would be stably maintained. We
confirmed the presence of the kanamycin-resistant trans-
poson insertion at the attTn7 site using PCR with primers
attF and attR (see Methods), which lie outside the attTn7
site (Fig. 3). Sequence analysis of five LVS and five Schu
clones determined that the insertion site occurs at either
25 bp (eight insertions) or 26 bp (two insertions) down-
stream of the glmS stop codon (data not shown), regardless
of strain. This is similar to the behaviour of Tn7 in P.
aeruginosa, in which the transposon inserts at two sites,
either 24 or 25 bp downstream of glmS (Choi et al., 2005).
In contrast, Tn7 inserts into a single site 25 bp downstream
of glmS in Y. pestis and E. coli (Choi et al., 2005; DeBoy &
Craig, 1996). Southern blot analysis using the aphA-1 gene
as a probe confirmed that there were no additional
insertions in the LVS chromosome (data not shown).
This is in agreement with the observations that transposi-
tion mediated by TnsABCD yields insertions only at attTn7
(Peters & Craig, 2001), and that Tn7 also confers
immunity, whereby it blocks transposition into a site
already occupied by a Tn7 element (DeBoy & Craig, 1996).
We used confocal microscopy to visualize the GFP in the
LVS Tn7 strain, but only ~10% of cells in each field
expressed GFP at one time (data not shown). We believe
E. D. LoVullo and others
1156 Microbiology 155
that a multitude of factors could have been responsible for
the poor visualization, including promoter strength,
improper folding of GFP, degradation and photobleaching.
After confirming the loss of the helper plasmid we tested
the cd-resolvase system. We transformed LVS and Schu
containing Tn7 insertions with plasmid pMP672 (Fig. 1d),
an unstable hygromycin shuttle vector expressing the cd-
resolvase from the F. tularensis blaB promoter. Select clones
were then grown in liquid media containing hygromycin
overnight and plated for single colonies on hygromycin
medium. These were then screened for loss of kanamycin
resistance, which occurred at a frequency of ~80% in both
LVS and Schu. Kanamycin-sensitive clones were cured of
the cd-resolvase plasmid in the same manner as the helper
plasmid. We then confirmed the loss of the kanamycin
marker with PCR, utilizing the Tn7 attF and attR primers
(Fig. 3). Sequence analysis of a resolved clone confirmed
that the two cd-res sites recombined into one cd-res site
with loss of the aphA-1 marker (data not shown).
Development of the blaB integration system
We have previously shown that LVS and Schu contain only
one functional b-lactamase, blaB (LoVullo et al., 2006).
The blaB gene lies in the Schu S4 chromosome with a
hypothetical gene transcribed in the opposite direction
435 bp upstream of its start site and a potential DNA/RNA
endonuclease family protein transcribed in the opposite
direction overlapping the blaB stop codon by 10 bp
(Larsson et al., 2005). Based on our sacB-based suicide
plasmid, we created a blaB integration vector that contains
1002 bp upstream of the blaB gene, a multiple cloning site,
and 593 bp downstream of the blaB gene that includes the
overlapping DNA/RNA endonuclease sequence.
The advantage of the blaB integration system is that we can
quickly screen for the clones with the unmarked insertion
in the secondary recombinant pool as they will be
ampicillin-sensitive and kanamycin-sensitive. This is in
contrast to the integration system developed for Francisella
novicida, which integrates into a gene that is present only in
the F. novicida chromosome and retains the kanamycin
selectable marker (Ludu et al., 2008).
Our blaB integration vector, pMP719 (Fig. 4a), is based on
the suicide vector pMP671, a pFNL10 Dori derivative of
pMP590 (LoVullo et al., 2006). To test the ability of the
system to integrate elements by allelic exchange, we cloned
lacZ under the Francisella rpsL promoter into the multiple
cloning site to form pMP790 (Fig. 4b). We selected the rpsL
promoter because we knew from our studies with the PrpsL–
gfp cassette that the promoter is active in E. coli (data not
Fig. 2. Tn7 system. A typical experimental procedure is performed by first electroporating the helper plasmid pMP720 into the
strain of interest and selecting for hygromycin-resistant transformants. One transformant is electroporated with the plasmid
containing the mini-Tn7 element (based on pMP749) containing your favourite gene (yfg), and transposon insertions are
selected on medium containing kanamycin. After curing a clone of the helper plasmid, the Tn7 insertion strain is ready for use. If
desired, the kanamycin marker can be deleted using the cd-resolvase plasmid pMP672.
F. tularensis integration systems
shown), and therefore allowed us to confirm b-galactosi-
dase production in E. coli before moving the system into F.
tularensis. We performed an allelic exchange experiment
similar to that shown in Fig. 5 for both LVS and Schu. We
confirmed the expression of lacZ from the chromosomes of
both strains by patching ampicillin-sensitive colonies onto
medium with X-Gal and observing blue colonies (data not
shown). We also confirmed the activity of the protein
produced in LVS by performing b-galactosidase assays. We
compared two DblaB::PrpsL–lacZ strains with wild-type
LVS. Three assays were performed on each strain: wild-type
LVS averaged 4 Miller units and the two integrated strains
averaged 300 Miller units, indicating expression of lacZ in
the novel location within the chromosome.
Improved sacB suicide plasmid and blaB
Our previously described sacB suicide vector, pMP590
(LoVullo et al., 2006), has proven itself useful for the
unmarked, in-frame deletions in both LVS and Schu, but
needed improvement (LoVullo et al., 2006). This allelic
exchange vector was developed from a shuttle vector that
was made non-replicative in F. tularensis by replacement of
the repA and ORF2 genes by the sacB gene, driven by the
repA promoter. However, the pFNL10 ori sequence is still
present in this plasmid, which could be problematic in
experiments that test gene essentiality by deleting a gene in
the presence of a plasmid carrying a wild-type copy of the
gene. In such an experiment, trans-acting replication
proteins from the plasmid could recognize the suicide
vector-borne ori sequence in the chromosome and initiate
replication that would likely be lethal due to incompat-
ibility with the natural chromosomal origin of replication.
To solve this problem, we removed the ori sequences and
repA promoter and inserted the F. tularensis dnaK
promoter region upstream of the sacB gene, which allowed
for strong expression of sacB such that the concentration of
sucrose in the selection medium could be reduced from 8
to 5%, while maintaining a very clean selection (data not
shown). This new plasmid was then subjected to inverse
PCR to remove 192 bp of DNA upstream of the dnaK
promoter region. This was done to prevent the integration
of the suicide vector into the chromosomal dnaK region,
which occurred often in test experiments (data not shown).
The final suicide vector, pMP812, is shown in Fig. 6(a).
Note that the dnaK promoter does not seem to be
recognized by E. coli, as pMP812 transformants are not
sensitive to sucrose. These improvements to our basic sacB
vector led us to modify our original blaB integration
vector, pMP719, in the same manner, resulting in pMP815
Fig. 3. pMP793 mini-Tn7 insertions into the attTn7 site of Schu. Genomic Schu DNA analysed by PCR using attTn7 outside
primers attF and attR. Lanes: 1, DNA marker; 2, pMP793 mini-Tn7 element (Tn7) inserted into the attTn7 of Schu with helper
plasmid present (3.8 kb); 3, Tn7 strain after curing the helper plasmid (3.8 kb); 4, Tn7 strain passaged in broth in the absence
of selection over 2 days (3.8 kb); 5, Tn7 strain transformed with the cd-resolvase plasmid pMP672 that has undergone site-
specific recombination of the cd-res sites (2.5 kb); 6, no-DNA control; 7, wild-type Schu (0.34 kb). We obtained similar results
E. D. LoVullo and others
1158 Microbiology 155
Fig. 4. Maps of the F. tularensis blaB integration vectors. (a) pMP719 is the first blaB integration vector based on the sacB-based suicide plasmid pMP671 that contains
1002 bp upstream of the blaB gene, a multiple cloning site, and then 593 bp downstream which includes the overlapping DNA/RNA endonuclease sequence. The BspHI site
may be used in conjunction with a restriction enzyme in the multiple cloning site to remove the putative blaB promoter. (b) pMP790 is a DblaB::PrpsL–lacZ integrating vector
based on integration vector pMP719. (c) pMP815 is a modification of pMP719 in which the repA promoter upstream of sacB has been replaced with a dnaK promoter from F.
F. tularensis integration systems
Fig. 5. blaB integration system. A typical allelic exchange procedure is done by electroporating the integration vector
containing your favourite gene (yfg) into the strain of interest and plating on medium containing kanamycin. Each electroporation
yields kanamycin-resistant recombinants at a frequency of 10”5–10”6relative to the transformation efficiency of our replicating
plasmids. The kanamycin-resistant primary recombinants are then screened for sucrose sensitivity. Kanamycin-resistant,
sucrose-sensitive primary recombinants are grown to saturation in medium lacking antibiotic and then plated onto sucrose
media to select against clones that do not undergo a second recombination event. Sucrose-resistant clones arise from cultures
at frequencies of 10”3–10”4relative to the viable counts. These putative secondary recombinants are then screened for loss of
kanamycin resistance and for ampicillin sensitivity as an indication that there is a successful recombination event and that
insertion of your element is likely. In the DblaB::PrpsL–lacZ experiment, ampicillin-sensitive colonies have ranged from 5 to 10%
of the population in LVS. The frequency of ampicillin-sensitive colonies is lower than expected, probably because of the
difference in length of the DNA flanking the DblaB allele (593 versus 1002 bp).
Fig. 6. Improved sacB suicide vector and
murI deletion. (a) Improved sacB vector
pMP812. The ori and repA promoter of
pMP590 were replaced with the Francisella
dnaK promoter to produce pMP780, and DNA
upstream of the dnaK promoter was removed
by PCR to produce pMP812. The multiple
cloning site (mcs) is also shown. A DmurI1
allele cloned within pMP812 was used to
delete the wild-type allele. (b) PCR amplicons
obtained from genomic LVS DNA with primers
specific to the murI region. Lanes: 1, DNA
marker; 2, wild-type LVS (2.7 kb); 3, LVS
DmurI1 strain PM2181 (2.0 kb).
E. D. LoVullo and others
Allelic exchange of murI and complementation
with the single-copy integration systems
To test these new tools, we sought to construct a strain
witha novel mutation,
complement it with a wild-type copy of the gene inserted
into the chromosome using each integration system. We
chose to interrupt the production of D-glutamate by
deleting the murI gene, which encodes a glutamate
racemase that is essential to E. coli (Doublet et al., 1993).
D-Glutamate is indispensable for the biosynthesis of
peptidoglycan in most eubacteria, and is produced through
two known routes: by D-amino acid transferase (D-AAT),
which converts a-ketoglutarate to D-glutamate by transa-
mination with D-alanine provided by the alanine racemase
reaction; and by glutamate racemase, which produces D-
glutamate through the racemization of L-glutamate (Liu
et al., 1998). In a number of bacteria, most notably in
certain Bacillus species, D-glutamate is also shuttled into
the production of poly-c-D-glutamic acid (PGA). In
Bacillus anthracis, there are two glutamate racemases
(RacE1 and RacE2) that produce D-glutamate for pepti-
doglycan as well as the biosynthesis of PGA, which
constitutes an antiphagocytic capsule, one of the two
major virulence factors of B. anthracis (Dodd et al., 2007;
Mock & Fouet, 2001). F. tularensis has capB and capC
genes, which share sequence homology at the amino acid
level of 38 and 29% with the CapB and CapC proteins of B.
anthracis that synthesize PGA (Su et al., 2007), and a
number of groups have shown that the capBC genes of F.
tularensis are important for tularemia pathogenesis (Maier
et al., 2007; Su et al., 2007; Weiss et al., 2007). However,
there is as yet no evidence that F. tularensis produces PGA.
Our inspection of the genomes of F. tularensis strains and
of F. novicida indicates that the gene FTT1197c, annotated
as murI, is the only glutamate racemase present in these
organisms, and we could find no genes encoding a D-AAT,
suggesting that there is only one source of D-glutamate in
these bacteria. However, a comprehensive transposon
library of the F. novicida genome has one mutant with an
insertion in the middle of the murI gene, and D-glutamic
acid was not included in the selection medium (Gallagher
et al., 2007). This might indicate that murI is dispensable.
To clarify this matter, we sought to determine whether a
murI deletion mutant would be auxotrophic for
glutamate, indicating the lack of any additional amino
acid racemase or D-AAT capable of compensating for the
loss of murI. We constructed an in-frame deletion of murI
in our improved sacB-based suicide vector, pMP812. Since
it has been shown that it is possible to rescue D-glutamate
auxotrophs with exogenous D-glutamate in Bacillus subtilis
and E. coli B/r and K-12 strains (Ashiuchi et al., 2007;
Doublet et al., 1993; Hoffmann et al., 1972), we performed
a standard two-step allelic exchange (LoVullo et al., 2006)
using pMP884 (Table 1) with D-glutamic acid present in
the medium. We then picked and patched 24 sucrose-
resistant secondary recombinants onto media with or
without D-glutamic acid. This yielded two recombinants
that could not grow on media lacking D-glutamic acid.
PCR was used to confirm that the secondary recombinants
auxotrophic for D-glutamic acid were murI deletions and
D-glutamic acid prototrophs were wild-type
recombinants (Fig. 6b). The murI deletion mutants formed
smaller colonies than the wild-type, suggesting a growth
defect. This is probably the reason why the phenotypes of
the secondary recombinants were skewed towards the wild-
type. One mutant, PM2181, was selected for further study.
We performed complementation studies on PM2181
utilizing the Francisella rpsL promoter driving murI in
the Tn7 system and the blaB system. The mini-Tn7 vector
pMP749 bearing PrpsL–murI+was introduced into PM2181
as shown in Fig. 2. The resulting attTn7::PrpsL–murI+
strain, PM2194, was prototrophic for D-glutamic acid. We
then resolved the kanamycin marker to ensure that it had
no effect on complementation of the murI lesion. As
expected, the resolved strain, PM2209, was able to grow in
the absence of D-glutamic acid. For the blaB system, a two-
step allelic exchange as shown in Fig. 5 was performed with
PM2210, was also able to grow in the absence of D-
glutamic acid. These results confirm that there is only one
Francisella. We do not know whether MurI also supplies
D-glutamate for PGA biosynthesis, but this mutant could
be used to identify such a polymer if it exists, since it
should be possible to label PGA with radioactive D-
glutamic acid supplied to PM2181 in culture.
biosynthesis in wild-type
The existence of a murI transposon mutant of F. novicida
(Gallagher et al., 2007) obtained without
supplementation suggests that there may be an extragenic
suppressor mutation in this mutant. To test this possibility
we performed suppressor analysis of our LVS D-glutamate
auxotroph. The strain was grown to saturation, washed, and
serially diluted on plates with and without D-glutamic acid.
We found that the strain produced suppressor mutants at a
frequency of ~161026per viable D-glutamic acid-requiring
colony-forming unit. This high frequency of suppression in
PM2181 supports the idea that the F. novicida transposon
mutant likely contains an extragenic suppressor.
We anticipate that these integration systems will be useful for
studies requiring single-copy gene expression, such as the
complementation of mutant genes when expression of the
wild-type gene from multi-copy
Furthermore, these systems will be helpful where the use of
multi-copy plasmids may not be suitable for cell culture or
animal experiments. They may also be helpful for developing
live vaccine strains containing additional antigens, where the
use of antibiotic-resistance markers is undesirable.
plasmids is toxic.
This work was supported by the Molecular Pathogenesis of Bacteria
and Viruses NIH grant T32 AI007362-18 to E.D.L., NIH grant
F. tularensis integration systems
AI068013 to M.S.P., and NIH grants AI058141 and AI065357 to
H.P.S. We wish to thank the members of the Pavelka lab for
reviewing this manuscript.
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F. tularensis integration systems