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Journal of Bacteriology, April 2001, p. 2384-2388, Vol. 183, No. 7
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.7.2384-2388.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
In Vitro Transposition System for Efficient
Generation of Random Mutants of Campylobacter
jejuni
Oscar R.
Colegio,1
Thomas J.
Griffin IV,2
Nigel
D. F.
Grindley,2 and
Jorge E.
Galán1,*
Section of Microbial Pathogenesis, Boyer
Center for Molecular Medicine, Yale School of Medicine, New Haven,
Connecticut 06536-0812,1 and Department
of Molecular Biophysics and Biochemistry, Yale University, New
Haven, Connecticut 06520-81142
Received 3 November 2000/Accepted 11 January 2001
 |
ABSTRACT |
Campylobacter jejuni is the most common cause
of food-borne illnesses in the United States. Despite the fact that the
entire nucleotide sequence of its genome has recently become available, its mechanisms of pathogenicity are poorly understood. This is in part
due to the lack of an efficient mutagenesis system. Here we describe an
in vitro transposon mutagenesis system based on the
Staphylococcus aureus transposable element
Tn552 that allows the efficient generation of insertion
mutants of C. jejuni. Insertions occur randomly and
throughout the entire bacterial genome. We have tested this system in
the isolation of nonmotile mutants of C. jejuni.
Demonstrating the utility of the system, six nonmotile mutants from a
total of nine exhibited insertions in genes known to be associated with
motility. An additional mutant had an inactivating insertion in sigma
54, implicating this transcription factor in flagellum regulation.
The availability of this efficient system will greatly facilitate the
study of the mechanisms of pathogenesis of this important pathogen.
| |
TEXT |
Campylobacter jejuni is
the most common cause of food-borne illnesses in the United States
(11). Despite its importance as a human pathogen and the
recent completion of the determination of the nucleotide sequence of
its genome (13), little is known about its mechanisms of
pathogenesis. This lack of knowledge is in part due to the lack of
suitable tools for the efficient generation of random mutants that can
be tested in relevant biological assays. Previous attempts to generate
mutants have relied on shuttle mutagenesis and homologous recombination
(2, 9, 19). More recently, an in vivo transposition system
based on the Himar1 transposable element has also been
reported (4). The weakness of this system, however, is
that restriction of the suicide vector is unavoidable, severely
affecting its efficiency. Although suitable for some applications,
these low-efficiency systems are less optimal when the isolation of a
large number of mutants is required. Difficulties in generating a
high-efficiency mutagenesis system in C. jejuni are most
likely due to the existence of powerful restriction barriers, inefficient expression of the appropriate transposase enzymes in vivo,
or a combination of these and other factors. To overcome these
limitations, we have designed an in vitro mutagenesis strategy based on
the Staphylococcus aureus transposon Tn552
(14, 15). This transposon exhibits features that resemble
those of Mu (12), such as the coding of a single-subunit
transposase and the requirement of a single accessory protein for in
vivo transposition (15). However, the ends of
Tn552, consisting of only 48-bp terminal inverted repeats
(10, 16), are much simpler than those of Mu and
Tn7. These are the only sequences required for
transposition, which facilitates the engineering of Tn552
for different applications. In contrast to Tn5
(5), Tn552 displays virtually no target preference (3, 6). A previous study has demonstrated
that Tn552 can insert efficiently and randomly into target
DNA molecules after in vitro transposition reactions (10).
These properties have allowed the use of this element as a tool for
nucleotide sequencing and mutagenesis (3, 6). We have
designed a derivative of Tn552 (Tn552kan-Campy)
that can be used in C. jejuni for the efficient generation
of random mutants. We have tested its performance in an in vitro
mutagenesis protocol to generate nonmotile mutants.
Construction of Tn552kan-Campy.
To construct a
derivative of Tn552 with an antibiotic resistance gene that
can confer resistance in C. jejuni, we replaced the
chloramphenicol resistance gene (cat) present in
Tn552cat (6) with the kanamycin resistance gene
aphA-3 from Campylobacter coli (18)
(Fig. 1). The Tn552cat-bearing
plasmid pTG426 (6) was digested with BsrGI,
which releases the cat cassette, and ligated to the
aphA-3 gene of pILL600 (8), yielding the
plasmid pSB1698, which carries Tn552kan-Campy. Since the
aphA-3 gene can also confer kanamycin resistance in
Escherichia coli, we compared the efficiency of
transposition of Tn552kan-Campy with that of Tn552cat. One hundred nanograms of pBluescript SKII plasmid
(Stratagene) DNA (as a target) was mixed with 100 ng (0.1 pmol) of
Tn552kan-Campy or Tn552cat DNA obtained from
pSB1698 or pTG426, respectively, by digestion with BglII and
subsequent agarose gel purification. The transposition reaction was
initiated by the addition of 30 ng of purified His-tagged TnpA
transposase and carried out for 1 h at 37°C in a total volume of
20 µl. The transposition reactions and the purification of TnpA were
carried out as previously described (6, 10). The reaction
mixtures were ethanol precipitated and electroporated into E. coli DH5
, and transposon insertions were selected by plating
the transformants on Luria broth plates containing either 50 µg of
kanamycin or 30 µg of chloramphenicol per ml to select for
Tn552kan-Campy or Tn552cat, respectively. A total
of 1.38 × 103 kanamycin-resistant colonies and 9.46 × 102 chloramphenicol-resistant colonies were obtained,
indicating that Tn552kan-Campy can undergo transposition in
vitro with an efficiency similar to that of the parent element
Tn552cat (equivalent results were obtained in several
repetitions of this experiment). In all cases, no antibiotic-resistant
colonies were obtained in the absence of the TnpA transposase in the
reaction mixture.
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FIG. 1.
Diagram of Tn552kan-Campy. Only relevant
restriction enzyme sites are shown. BglII cleavage creates
the CAOH-3' ends needed for transposition and leaves a
5'-GATC single-stranded extension. L, left; R, right.
|
|
Generation of Tn552kan-Campy insertion mutants of
C. jejuni.
We next tested the use of
Tn552kan-Campy in the generation of random insertion mutants
in C. jejuni. Chromosomal DNA was isolated from the C. jejuni strain 81-176 by using Qiagen (Qiagen, Inc., Valencia,
Calif.) according to the manufacturer's instructions. One hundred
nanograms of C. jejuni DNA was mixed with 100 ng (0.1 pmol)
of agarose gel-purified Tn552kan-Campy DNA, and the
transposition reaction was carried out as described above. The reaction
mixture was ethanol precipitated and electroporated into C. jejuni 81-176 as previously described (8), and
kanamycin-resistant colonies were selected on brain heart infusion
(BHI) agar plates containing 30 µg of kanamycin per ml. Approximately
100 kanamycin-resistant colonies were recovered in several repetitions
of this experiment, which represents a significant drop from the number
of transposition events obtained using an E. coli plasmid as
a target (see above). The reduction in the number of
Tn552kan-Campy insertion events obtained with C. jejuni can be partially attributed to the requirement of
homologous recombination for the rescue of kanamycin-resistant colonies
(see below). However, we hypothesized that, most likely, DNA
restriction of the E. coli-grown Tn552kan-Campy
DNA was responsible for a significant portion of the reduction in the
number of insertion events recovered in C. jejuni. We tested
this hypothesis by constructing a C. jejuni shuttle plasmid
carrying Tn552kan-Campy. This plasmid was constructed by
cloning the 1.5-kb SpeI fragment from pSB1698 carrying
Tn552kan-Campy into the unique SpeI site of the
C. jejuni shuttle plasmid pRY112 (8). The
resulting plasmid, pSB1699, was introduced into C. jejuni
strain 81-176 by conjugation. Plasmid pSB1699 was isolated from
C. jejuni 81-176 and digested with BglII to
liberate Tn552kan-Campy. One hundred nanograms of C. jejuni 81-176 genomic DNA was mixed with 100 ng of
Tn552kan-Campy DNA obtained from pSB1699 grown in C. jejuni 81-176, and the transposition reaction was carried out as
described above. After electroporation of C. jejuni 81-176,
between 2.85 × 103 and 7.68 × 103
kanamycin-resistant colonies were recovered per reaction in different independent experiments. In all cases, the absence of the TnpA transposase from the reaction mixture resulted in no
antibiotic-resistant colonies after electroporation. The significant
increase in the number of kanamycin-resistant colonies obtained using
Tn552kan-Campy isolated from C. jejuni indicates
that the low yield of insertions obtained with the E. coli-grown transposon DNA is probably the consequence of DNA
restriction barriers between these two microorganisms.
Tn552kan-Campy insertions occur throughout the C. jejuni chromosome.
Tn552 has been reported to
insert nearly randomly in plasmid targets (3, 6). To
determine if Tn552kan-Campy inserted throughout the C. jejuni genome, Southern blot analysis of 16 randomly chosen
kanamycin insertion mutants was carried out using purified
Tn552kan-Campy DNA labeled with a randomly primed
nonradioactive labeling kit (ECL RPN 3040; Amersham) as a probe.
Chromosomal DNAs from the different C. jejuni strains were
isolated as indicated above, digested with BspHI, separated
on a 0.9% agarose gel, and transferred to a GeneScreen Plus
membrane (NEN Life Science Products) according to standard procedures
(17). BspHI was chosen because it cuts within
the transposon. Therefore, when probed with
Tn552kan-Campy DNA, the digested chromosomes were expected
to produce two unique bands of different sizes if the different mutants
were the result of a single insertion event. As shown in Fig.
2, all but one sample showed two unique
bands representing unique transposon insertions. One sample produced a
single band that additional experiments demonstrated to be the result
of the overlap of two bands of similar sizes (data not shown). As
expected, the wild-type C. jejuni did not show hybridization
to the Tn552kan-Campy probe. These results indicate that the
Tn552kan-Campy insertions are distributed throughout different sites of the C. jejuni chromosome.
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FIG. 2.
Southern blot analysis of Tn552kan-Campy
insertions in C. jejuni. Chromosomal DNAs from randomly
chosen insertion mutant strains were digested with BspHI,
separated on an agarose gel, transferred to a GeneScreen Plus membrane,
and probed with labeled Tn552kan-Campy DNA. R1 to R16,
C. jejuni Tn552kan-Campy insertion mutants; wt,
wild-type C. jejuni 81-176.
|
|
To investigate if Tn
552kan-Campy exhibits any preference
towards certain regions of the
C. jejuni chromosome, we
determined
the precise sites of insertion of several randomly chosen
mutations
by nucleotide sequencing. Genomic DNAs from the different
C. jejuni insertion mutant strains were prepared as follows.
Approximately
10
10 C. jejuni cells were scraped
from BHI plates and resuspended
in 567 µl of TE (10 mM Tris [pH
8.0], 1 mM EDTA). Bacterial cells
were lysed by adding 30 µl of 10%
sodium dodecyl sulfate and 3
µl of 20-mg/ml proteinase K and
incubating the cell suspension
at 37°C for 1 h. Cell lysates
were treated with 100 µl of 5 M
NaCl and 80 µl of CTAB-NaCl
solution (5% hexadecyltrimethylammonium
bromide [CTAB], 0.5 M NaCl)
and incubated for 10 min at 65°C.
Cell lysates were then extracted
once with chloroform, six times
with phenol-choloform-isoamyl alcohol
(25:24:1), once with chloroform,
and twice with ether. DNA was ethanol
precipitated and resuspended
in 150 µl of TE. Ten micrograms of
genomic DNA from each mutant
was digested overnight with
BspHI, ethanol precipitated, and resuspended
in 11 µl of
TE. Genomic DNA sequences were then determined using
an ABI PRISM
BigDye Terminator Cycle Sequencing Ready Reaction
Kit (Applied
Biosystems, Perkin-Elmer Corporation) according to
the recommendations
of the manufacturer and with a primer directed
outward from the 5' end
of the
aphA-3 kanamycin resistance gene
(5'-CCAATTCTCGTTTTCATACC-3'). The sequence obtained from
each
of the mutants was compared to the nucleotide sequence of the
C. jejuni NCTC 11168 chromosome (
14). As shown
in Table
1 and
Fig.
3, all insertions analyzed occurred at
different sites of
the
C. jejuni genome and showed no
clustering in any particular
area of the chromosome.
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FIG. 3.
Diagram of the C. jejuni chromosome showing
the positions of the different Tn552kan-Campy insertions.
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|
Isolation of C. jejuni nonmotile mutants using
Tn552kan-Campy.
To test the utility of the
Tn552kan-Campy in vitro transposition system, we screened a
panel of in vitro-generated transposon insertion mutants for motility
defects. We chose motility as a testing phenotype because several genes
involved in this phenotype are well characterized and known to be
located throughout the C. jejuni genome (7,
19). Nonmotile Tn552kan-Campy insertion mutants of
C. jejuni strain 81-176 were identified in semisolid BHI
agar (containing 37 g of BHI and 4 g of agar per liter of medium). Of 205 kanamycin-resistant mutants screened, 9 exhibited defects in motility. To identify the mutated genes, the transposon insertion sites were determined by sequencing analysis as described above. Six of the nine nonmotile mutants revealed transposon insertions in genes known or expected to be associated with motility (Table 2). The two remaining mutants had
insertions in genes of unknown function not previously associated
with motility. These results confirmed the usefulness of the
Tn552kan-Campy in vitro transposition system for the
isolation of random mutants.
Analysis of Tn552kan-Campy insertions.
In the
system described here, interpretation of the phenotypes of
transposon-promoted mutations depends on the assumptions that the
recovered insertions do not contain gross rearrangements of the target
DNA (such as large deletions) and that transposon-disrupted genes are
incorporated by homologous recombination, replacing the wild-type
chromosomal locus. Our expectation of homologous recombination was
confirmed by PCR using primers complementary to chromosomal
sequences bracketing the insertion site. In all six cases tested,
the band corresponding to the preinsertion wild-type locus was lost and
was replaced by a new band that was larger by ~1.3 kb, which is the
size of the transposon (data not shown).
DNA sequence analysis of the transposon-target junctions of 19 insertions (Fig.
4) showed that all were the result of
simple
concerted insertions at a single target site; none were
accompanied
by unexpected rearrangements such as deletions and
inversions.
However, target junctions were unusual and indicated that
C. jejuni processes the gapped products of in vitro
transposition very differently
from
E. coli. Only 3 of the
19 insertions were flanked by a simple
target duplication of the type
seen previously with many hundreds
of in vitro Tn
552
insertions recovered in
E. coli (
6). In most
of
the
C. jejuni insertions, vestiges of a target duplication
were observed, but in addition one junction contained from 1 to
4 bases
derived not from the target sequence but from the 5'-GATC
that extended
from the 5' end of the transposon substrate DNA.
In three cases a
portion (3 or 4 bases) of this GATC sequence
had completely substituted
for the target duplication.
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FIG. 4.
Target site sequences of Tn552kan-Campy in
C. jejuni 81-176 genomic DNA. Junction sequences were
determined using outward primers complementary to the
aphA-3-resistant gene located within
Tn552kan-campy (5' end, 5'-CCAATTCTCGTTTTCATACC-3';
3' end, 5'-GGATCAAGCCTGATTGGGAG-3'). Capital letters
indicate the Tn552kan-Campy transposon, lowercase letters
indicate the C. jejuni genomic DNA sequence, outlined
letters indicate added nucleotides, and boldface letters indicate
target site repeats.  , a single C is missing at the site of
insertion.
|
|
In vitro, the Tn
552 transposase joins the
CA
OH-3' transposon end to target DNA (attacking
phosphodiester bonds on each DNA
strand that are separated by a 6- to
9-base 5' stagger), giving
a product with 6- to 9-base gaps at
each transposon target junction.
Preexisting single-strand extensions
on the 5' ends of the transposon
substrate (5'-GATC for the substrates
used here) are unaffected
by transposase in vitro and are
efficiently removed in
E. coli (probably by the 5'
"flap" exonuclease of DNA polymerase I) during
the gap repair
and ligation process. The most straightforward
explanation for the
retention of the 5' flank in
C. jejuni (and
probable loss of
some of the target duplication) is that it uses
microhomologies to pair
within the single-strand gap region and
become ligated to the 3' end of
the duplex as repair synthesis
proceeds.
While these anomalous target junctions have no affect on the utility of
the transposon mutagenesis, their formation may be
prevented by
processing the gapped intermediate with DNA polymerase
I (with
deoxynucleoside triphosphates) in vitro before electroporation
of
C. jejuni.
Conclusions.
We have described an in vitro transposition
system for the generation of random insertion mutants of
C. jejuni. Unlike previously described systems,
the Tn552-based system is very efficient, generating up to ~8,000 mutants in a single reaction using 100 ng of target DNA.
The efficiency of this system combined with the simplicity of the
design of the element will allow the easy construction of derivatives
suitable for signature-tagging mutagenesis, the isolation of in
vivo-expressed genes, and random PhoA or LacZ protein fusions. In
addition, the development of an in vitro transposition system combined
with the availability of the entire nucleotide sequence of the C. jejuni genome will allow the application of specialized
mutagenesis protocols to identify virulence as well as essential genes
(1).
| |
ACKNOWLEDGMENTS |
We thank M. Blaser and P. Guerry for bacterial strains and plasmids
and María Lara-Tejero for critical review of the manuscript and
experimental advice.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Section of
Microbial Pathogenesis, Boyer Center for Molecular Medicine, Yale
School of Medicine, New Haven, CT 06536-0812. Phone: (203) 737-2404. Fax: (203) 737-2630. E-mail: jorge.galan{at}yale.edu.
| |
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Journal of Bacteriology, April 2001, p. 2384-2388, Vol. 183, No. 7
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.7.2384-2388.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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