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Infection and Immunity, January 2000, p. 415-419, Vol. 68, No. 1
0019-9567/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Isolation of Bacteroides fragilis Mutants with In Vivo
Growth Defects by Using Tn4400', a Modified
Tn4400 Transposition System, and a New Screening
Method
Yixin P.
Tang and
Michael H.
Malamy*
Department of Molecular Biology and
Microbiology, Tufts University School of Medicine, Boston,
Massachusetts 02111
Received 5 March 1999/Returned for modification 14 May
1999/Accepted 5 June 1999
 |
ABSTRACT |
A modified version of the Bacteroides fragilis
transposon Tn4400, designated Tn4400', enabling
rapid isolation and analysis of B. fragilis mutants has
been constructed. To identify potential virulence factors,
Tn4400'-generated mutants were screened by a new method;
this resulted in the isolation of 21 mutant strains with impaired
growth characteristics on tissue culture monolayers but normal growth
in rich medium anaerobically.
| |
TEXT |
Tn4400' mutagenesis.
Bacteroides fragilis is
the most frequently isolated anaerobic species from human
intraperitoneal and intra-abdominal infections involving anaerobes
(2, 3, 9). Compound transposon Tn4400 was
isolated from B. fragilis plasmid pBFTM10 by Robillard et al. (7). A similar transposon, Tn4351, studied in
the laboratories of Smith (11) and Salyers (5,
10), was used to generate B. fragilis and
Bacteroides thetaiotaomicron mutants. Here we describe
further modifications of Tn4400 which have increased its
transposition frequency as well as facilitated the rapid isolation of
B. fragilis chromosomal fragments abutting the inserted transposon.
The entire transposon Tn4400, including a
clindamycin-resistant (Clnr) determinant active in B. fragilis, was cloned as a single BglII fragment from a
pBFTM10-F' lac fusion plasmid, pOX446R1 (7), into
a plasmid based on pDG5, which contains the pBR322 replicon, the
bla gene for ampicillin resistance (Ampr) in
Escherichia coli, and the origin of transfer
(oriT) from the IncP plasmid RK2 (4). The
resulting plasmid, pNJR609, apparently underwent an
oriT-mediated rearrangement upon prolonged storage and
consequently lost the oriT region. This plasmid was named pNJR609
(Table 1).
To restore the ability of the plasmid to be mobilized by RK2, the
oriT of RP4 was isolated as a blunt-ended 760-bp
HaeII fragment
from pJST51 (
14) and cloned into
the
HincII site within the
bla gene of
pNJR609
. The resulting plasmids, pYT644A and pYT644B,
were able to
generate Cln
r colonies by transposition upon transfer into
B. fragilis. A
tetQ gene cassette, which confers
tetracycline resistance in
B. fragilis but not
E. coli (
6), was cloned as a
SacI fragment into
a pSP72
vector plasmid, generating pRG23. A
BglII-
BamHI fragment from
pRG23 containing the
tetQ gene was cloned into the
BglII sites
of
pYT644A and pYT644B. The resulting plasmids, pYT645A and pYT645B
(Fig.
1A), were able to generate both
Cln
r and tetracycline-resistant (Tet
r)
B. fragilis strains. For conjugal transfer of the pYT644 or
pYT645
plasmid from
E. coli to
B. fragilis,
mid-log-phase broth
cultures of an
E. coli donor strain, a
second
E. coli strain containing
the mobilizer RK231, and a
B. fragilis recipient were mixed (2
ml-2 ml-5 ml) and
concentrated by centrifugation, and the mixture
was placed on a sterile
filter (type HAWP 047; Millipore Corp.,
Bedford, Mass.) on the surface
of a plate containing solidified
brain heart infusion broth
supplemented with 0.5% yeast extract
and 5 µg of hemin per ml
(BHIS). After overnight, aerobic incubation
at 37°C, the bacteria
were plated on BHIS plates containing gentamicin
(50 µg/ml), rifampin
(50 µg/ml), and tetracycline (2 µg/ml) or
clindamycin (6 µg/ml).
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FIG. 1.
Schematics of pYT645 (A) and pYT646 (B), the
Tn4400' transposon delivery plasmids for B. fragilis. The tetX gene confers resistance to
tetracycline only in aerobically grown E. coli
(12). Parts of these plasmids have not yet been sequenced,
and some of the endonuclease recognition sites may deviate slightly
from the precise positions shown here. (L), left; (R), right.
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To expedite isolation of DNA flanking the inserted transposon, a 1.6-kb
PstI fragment from pJST51, which contains the 5' half
of the
bla gene, was cloned into the
PstI sites of
pYT645A and
pYT645B. The resulting Amp
r plasmids, pYT646A
and pYT646B (Fig.
1B), were used for large-scale
mutagenesis of TM4000,
originally a clinical isolate and the standard
strain used in our
pathogenesis studies. The two versions of pYT646
differ only in the
orientation of RK2
oriT. The inverse transposon
on pYT646 is
named Tn
4400'. A
B. fragilis chromosome
mutagenized
by an inverse transposition event is depicted in Fig.
2A.
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FIG. 2.
The B. fragilis chromosome after
Tn4400' insertion and the clone-out strategy. (A) After
Tn4400' insertion, the B. fragilis cell becomes
Tetr and Clns. The letters a through f
designate arbitrary markers in the B. fragilis chromosome.
The number 1 indicates oriT from RP4; the number 2 indicates
rep from pBR322. (B) Schematic of the cloning of B. fragilis chromosomal fragments adjacent to Tn4400'. The
Tn4400'-containing B. fragilis chromosome is
digested with BclI. Self-ligation of the
Tn4400'-containing BclI fragment generates an
Ampr E. coli plasmid. (C) Schematic of the
clone-out technique. Tn4400'-containing B. fragilis chromosomal DNA is cut with HindIII and
religated to transform E. coli to Ampr. Plasmids
purified from these Ampr colonies contain the right-hand
half of Tn4400' plus a fragment of B. fragilis
chromosomal DNA. When a high concentration of chromosomal fragments is
used in a clone-out ligation, the resulting plasmids may contain more
than one HindIII fragment. This causes little difficulty
because the additional HindIII sites will appear in the
sequencing results. The multiple HindIII fragments
contained in the same clone-out plasmid usually come from different
loci on the B. fragilis chromosome (data not shown). (R),
right; (L), left.
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In
B. fragilis the inverse transposition events occurred
more frequently than direct Tn
4400 transposition or
cointegrate formation
events (Table
2).
(Curiously, pYT645A and pYT645B, which differ
only in the orientation
of RK2
oriT, gave different ratios of
Tet
r to
Cln
r colonies.) A typical conjugation experiment with
HB101/pYT646B
as the donor gave rise to Tet
r and
clindamycin-sensitive (Cln
s) mutants at a frequency between
10
7 and 10
6 per input donor cell. Since the
conjugation experiments were
done aerobically and
B. fragilis does not replicate under these
conditions, many
transposon mutants could be isolated from one
single mating. Among 50 Tn
4400'-generated mutants analyzed in
detail, only three
(5%) were siblings.
It is relatively simple to "clone out" the chromosomal fragment
adjacent to the inverse transposon, as shown in Fig.
2. The
mutant
chromosome can be cut with
HindIII, and the fragments
religated
to transform
E. coli, selecting for
Amp
r colonies. Alternatively, when a
HindIII
site occurs in the
B. fragilis chromosome very close to the
right end of Tn
4400', the
mutant chromosome can be cut with
PstI and the resulting fragments
can be ligated with the
1.6-kb
PstI band from pJST51 before being
transformed into
E. coli with selection for Amp
r colonies. The
ligation regenerates a complete
bla gene. Chromosomal
sequences abutting the left end of Tn
4400' are more
difficult
to obtain. The mutant chromosomal DNA can be cut with
BclI, which
does not cut within the entire
Tn
4400' transposon, and then religated
and used to transform
E. coli to Amp
r. The resulting plasmid recovered
from
E. coli will contain chromosomal
DNA flanking both
transposon ends (Fig.
2B). The clone-out technique
is unsuitable if the
mutant is a Tet
r Cln
r cointegrate. However,
very few (4%) of the Tet
r colonies generated by pYT646
were cointegrates. When pYT645 was
used as the mutagen, the frequencies
of cointegrate formation
were slightly higher (Table
2).
Since Tn
4400' is a transposon designed for generalized
mutagenesis, its target sequence specificity is of great importance.
Tn
4400' apparently has some preference for AT-rich regions
(data
not shown). However, its insertion sites are numerous on the
B. fragilis chromosome. The sequences of more than 50 separate mutants
have been analyzed, and except for 3 which are likely
to be siblings
(since they were isolated from the same mating), no two
mutants
had Tn
4400' inserted in the same place. Mutants were
also screened
for the presence of auxotrophs. The percentages of
auxotrophs
among the Tn
4400' mutants obtained from each
mating were consistently
slightly above 1%, regardless of whether
pYT646A or pYT646B was
used for the mutagenesis. Overall, a total of
more than 11,000
mutants were screened this way, and 1.2% of the
mutants were found
to be
auxotrophs.
Under the experimental conditions just described, most
B. fragilis mutants received only one copy of the inverse transposon.
The results of Southern blot analysis of 12 Tet
r
Cln
s mutants are shown in Fig.
3. Southern hybridization was
carried
out with an ECL Direct kit (Amersham Life Science, Little
Chalfont,
Buckinghamshire, England). Plasmid pYT646B was used to
make probes.
Two mutants, YT58.1.3 and YT129.2.20, appeared to contain
more
than one copy of Tn
4400'. In one of the mutants,
JD3a.3, the Tn
4400'
transposon appeared to have undergone
rearrangement, with the
loss of part of its sequence.
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FIG. 3.
Southern blot analysis of the chromosomes of 12 Tetr Clns mutants. The entire pYT646B plasmid
was used to generate hybridization probes. (A) Southern blot analysis
after complete HindIII digestions of the mutant
chromosomal DNAs. The arrow indicates the fragment internal to
Tn4400'. Each copy of Tn4400' inserted in a
mutant chromosome will give rise to two junction fragments. Mutant
YT129.2.20 appears to contain two copies of Tn4400'. The
transposon in the chromosome of mutant JD3a.3 has undergone
rearrangement. (B) Southern blot analysis after complete
PstI digestions. The arrow indicates the PstI
fragment internal to Tn4400' (as defined by the structure of
pYT646). The DNA from mutant YT58.1.3 clearly showed more than two
junction fragments. YT58.1.3 may have suffered more than one
transposition event.
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|
In agreement with previous reports on the closely related
Tn
4351 (
5,
11), Tn
4400' transposition
resulted in a 3-bp duplication
at the target site (data not shown).
Tn
4400' insertion may also
cause deletions at the target
site. However, the transposon apparently
did not cause mutations in
unrelated sites in the bacterial chromosome
at a high frequency. To
date, eight transposon mutants with discernible
phenotypes have been
complemented with
B. fragilis chromosomal
DNA fragments. All
of the complementing fragments overlap the
regions of the chromosome
where Tn
4400' insertions occurred in
the corresponding
mutants.
Once the inverse transposon is in the chromosome, it appears to be very
stable: reversion events do not occur at any observable
frequency. A
number of auxotrophs were isolated after Tn
4400'
mutagenesis, but none reverted to prototrophy at frequencies greater
than 10
9. Occasionally, Tet
s derivatives of
Tn
4400' mutants were isolated, but the mutant
phenotypes
were not reversed. Apparently, internal portions of
the inverse
transposon, including the
tetQ gene, were
lost.
Screen for monolayer growth-deficient (MGD) mutants.
The
tissue culture monolayer system for B. fragilis
pathogenecity (1a) was used to screen for mutants which grow
poorly on animal cell monolayers incubated aerobically but which grew normally in an anaerobic chamber. These mutants may be defective in
virulence factors such as aerotolerance, attachment to animal cells,
and interaction between the bacteria and the animal cells.
It was empirically determined that, when a mixture of sodium dodecyl
sulfate and
p-nitrophenyl-conjugated substrate for
N-acetyl-
-
D-glucosaminidase
(NAGase) was
added, infected Monika cell (a murine fibroblast
cell line) monolayers
produced a much more intense yellow color
than uninfected monolayers.
Moreover, when monolayers were infected
with TC2, a
B. fragilis mutant that grows poorly on tissue culture
monolayers
(
8), the yellow color was not as intense as when
wild-type
B. fragilis strains were used. These observations led
to the
development of the enzyme assay into a rapid screen for
MGD
B. fragilis mutants, although it is not known whether the
NAGase
activity detected on infected monolayers was a result of
enzyme
production by the bacteria or heightened enzyme production
by the
animal cells in response to the bacterial
infection.
Monika cells were passaged as previously described (
1), with
the exception that 8% (vol/vol) fetal bovine serum (FBS; HyClone,
Logan, Utah), instead of 10% FBS, was used in the medium. Confluent
monolayers were diluted 1:5 and seeded in 24-well tissue culture
plates
(Corning Costar, Cambridge, Mass.) and incubated for 2
days before
bacteria were introduced. Individual Tn
4400'-containing,
Tet
r B. fragilis candidates were picked with
sterile toothpicks and
transferred onto a BHIS plate containing 2 µg
of tetracycline
per ml, and the same toothpicks were washed in wells of
a 96-well
plate, each containing 100 µl of MPBS buffer (0.01 M sodium
phosphate
buffer [pH 6.9], 0.85% NaCl, 0.1% gelatin). Fresh RPMI
1640 medium
with 8% FBS was added to confluent Monika cell monolayers
in 24-well
tissue culture plates at 0.5 ml/well, along with 5 µl of
the bacterial
suspension. One well per plate was not inoculated with
bacteria
as a negative control. The infected monolayers were incubated
at 37°C under a normal atmosphere containing 5% CO
2 for
48 h.
The supernatant in the wells was then aspirated, which left
behind
the animal cell monolayers and attached bacterial cells. A
0.5-ml
aliquot of a substrate mixture containing 3 mM
p-nitrophenyl
N-acetyl-
-
D-glucosaminide
(N9376; Sigma, St.
Louis, Mo.), 0.2 M sodium phosphate buffer
(pH 6.0), and 0.5% sodium
dodecyl sulfate was added to each well.
After the plates were incubated
at 37°C for 1 h, 1 ml of 1 M sodium
carbonate was added to each
well and the plates were inspected
by
eye.
One advantage of this screen lies in the fact that it was not necessary
to keep the bacterial inocula constant to distinguish
a
growth-deficient mutant from a growth-competent mutant. For
a
growth-competent strain, initial inocula ranging from 10
3
to 10
7 viable cells per well would generate yellow colors
of approximately
the same intensity. Mutants with even a small defect
in growth
on tissue culture monolayers would generate a lighter color
even
when they were inoculated at 10
6 viable cells per
well.
A total of 7,222 individual Tn
4400'-generated mutants were
screened; 44 strains persistently generated lighter colors in the
screen. These candidates were tested for growth in rich medium
in the
anaerobic chamber, and about half of the strains were determined
to be
slow growers. The remaining mutant candidates were seeded
onto Monika
cell monolayers in 60-mm-diameter tissue culture dishes,
and growth
curves were determined. Twenty-one mutants showed defects
when they
grew on tissue culture monolayers but not in rich medium
in the
anaerobic chamber. These mutants were termed MGD
mutants.
Four mutants were found to have normal growth kinetics when their
growth on monolayers was measured directly. However, during
the NAGase
screen, the wells containing these four mutants had
lighter color.
These four mutants were termed the pale mutants.
Wells containing two
of the four pale mutants were slightly less
yellow during the assay,
whereas wells containing the remaining
two were almost
colorless.
The MGD mutants were subjected to the clone-out analysis. Potential
open reading frames in the vicinity of the transposon
insertions were
analyzed by homology. Several of the disrupted
open reading frames may
potentially code for proteins with remarkable
similarity to proteins of
known functions from other organisms.
For example, the disrupted gene
in mutant YT22.1.22 codes for
a protein with similarity to the LysA
protein of
Pseudomonas species.
This mutant was confirmed to
be a lysine
auxotroph.
However, most of the disrupted potential open reading frames have no
matches to proteins of known function in the databases.
Three
mutants, YT65.2.10, YT129.2.20, and YT135.2.8, when exposed
to
atmospheric oxygen were found to be less aerotolerant than
TM4000 (data
not shown). All three mutants have been complemented
by a
B. fragilis chromosomal fragment library. The complementing
fragment
for YT135.2.8 contains five open reading frames, which
constitute the
batI locus (
13).
One mutant, YT58.1.3, showed a decreased level of attachment to
glutaraldehyde-fixed Monika cell monolayers. A DNA fragment
containing
an open reading frame disrupted in this mutant, named
the
bafA gene, was amplified from the wild-type
B. fragilis chromosome
by PCR. This fragment, when introduced into
YT58.1.3, enhanced
its binding properties, as well as its growth on
animal cell monolayers
(our unpublished
data).
Conclusion.
Transposons are widely used for general
mutagenesis of bacteria. The improved transposon mutagen described
above, Tn4400', has several useful properties which make it
an ideal reagent for mutagenesis of B. fragilis. Its
insertion into the B. fragilis chromosome produced a large
set of different mutants. By the aerobic mating protocol, most of the
mutants were not siblings. Moreover, multiple insertions were not
common. The insertion events were not readily reversible, which
therefore obviated the need to constantly apply antibiotic pressure to
retain the transposon. The ease of isolation of the DNA sequences
flanking the insertions has made possible DNA sequence analysis of many
MGD mutants. Furthermore, preliminary results showed that
Tn4400' insertion events can be detected in other
Bacteroides strains (M. M. Dallas, M. F. Maiden, Y. P. Tang, M. J. Duncan, and M. H. Malamy, unpublished
data) and in Porphrymonas gingivalis (1).
| |
ACKNOWLEDGMENTS |
The research reported in this paper was supported by grant AI-19497
from the National Institutes of Health.
We are grateful to B. E. B. Claesson for providing the Monika
cell line, A. A. Salyers for providing the plasmid containing tetQ, M. M. Dallas for performing the screen for
auxotrophs in Tn4400'-mutagenized B. fragilis,
R. A. Gallegos for providing plasmid pRG23, and J. DePonte III for
isolating B. fragilis strains auxotrophic for arginine.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Biology and Microbiology, Tufts University School of
Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-6756. Fax: (617) 636-0337. E-mail: mmalamy1{at}opal.tufts.edu.
Editor:
J. T. Barbieri
| |
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Infection and Immunity, January 2000, p. 415-419, Vol. 68, No. 1
0019-9567/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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