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Journal of Bacteriology, October 2000, p. 5391-5398, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Mutagenesis of Neisseria meningitidis by
In Vitro Transposition of Himar1 mariner
Vladimir
Pelicic,1,*
Sandrine
Morelle,1
David
Lampe,2 and
Xavier
Nassif1
INSERM U411, Laboratoire de Microbiologie,
Faculté de Médecine Necker-Enfants Malades, 75015 Paris, France,1 and Department of
Biological Sciences, Duquesne University, Pittsburgh, Pennsylvania
152192
Received 22 May 2000/Accepted 10 July 2000
 |
ABSTRACT |
Now that the meningococcal genome sequence has been completed, the
lack of a suitable method for saturation mutagenesis remains a major
obstacle to the unraveling of the pathogenic propensity of
Neisseria meningitidis. Here, we demonstrate that in vitro Himar1 mariner transposition on chromosomal or
PCR-amplified meningococcal DNA, which is subsequently reintroduced
into N. meningitidis by natural transformation, is an
extremely efficient mutagenesis method. Southern blot analysis,
sequencing the Himar1 insertion point in numerous
transposition mutants, and a limited screening of the mutant libraries
for clones impaired in maltose catabolism confirmed that
Himar1 transposed randomly in N. meningitidis. Taken together, these data demonstrate that Himar1 in vitro
transposition can lead to the exhaustive mutagenesis of N. meningitidis, allowing for the first time a genomic-scale
mutational analysis of this important human pathogen.
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INTRODUCTION |
With the completion of the genomic
sequences of two different strains, Z2491 (24) and MC58
(38), Neisseria meningitidis entered the
postgenomic era (35). The first example of the utility of
such a sequence was the in silico identification of several promising
vaccine candidates against serogroup B meningococci (28) for
which no vaccine is available. It is very likely that further
deciphering of its genomic content will lead to a better comprehension
of the genetic determinants essential for the meningococcus peculiar
properties. However, as already noted for other bacteria whose genomes
have been sequenced, almost half (46.3%) of the identified genes in
MC58 (38) encode proteins with no predictable function. In
addition, some of the functions assigned on the basis of sequence
homologies may turn out to be erroneous. Therefore, to convert sequence
data into meaningful biological information, two kinds of mutagenesis
procedures are required: one allowing the rapid mutagenesis of defined
genes for the identification of the linked phenotypes, and one allowing
random mutagenesis of the whole genome for the identification of the
genes responsible for defined phenotypic traits.
Unfortunately, none of the presently available mutagenesis methods
seems usable as such for a genomic-scale mutagenesis of N. meningitidis. Indeed, although facilitated by the fact that the
meningococcus is naturally transformable, allelic exchange mutagenesis
remains mostly a gene-by-gene analysis method. Unless a high-throughput
strategy involving numerous laboratories is envisioned, as for the
budding yeast Saccharomyces cerevisiae (42),
allelic exchange seems impracticable for a saturation mutagenesis of
N. meningitidis. For example, mutagenesis of the 2,230 predicted open reading frames (ORFs) in the genome of Z2491 (24) would require at least 4,460 cloning steps (one step to clone each gene and another one to introduce a resistance cassette within each of them). Moreover, some genomic regions are known to be
refractory to cloning in Escherichia coli and would
therefore be difficult to mutagenize. Therefore, transposon mutagenesis seems to be the method of choice. However, conventional transposon mutagenesis has been limited in Neisseria mostly due to the
lack of suitable neisserial transposons. Moreover, derivatives of
well-known E. coli mobile elements, such as Tn5,
are not active in Neisseria (39). Consequently,
conjugative transposons and shuttle mutagenesis have been used to
create mutant libraries in Neisseria. Conjugative transposons like Tn916 (9) are large elements
that are self-transferable by conjugation to a variety of bacteria,
where they integrate into the chromosome by transposition. When
introduced into N. meningitidis, Tn916
(15) and a Tn916-like derivative (21) transposed into different sites, as evidenced by the identification of
mutants presenting precise genetic defects (8, 34, 37). Conjugative transposons are, however, not practical for saturation mutagenesis of N. meningitidis for several reasons: (i) the
frequency of transposition is low, and thus the number of mutants falls far short of the number statistically required for the mutagenesis of
all N. meningitidis genes; (ii) the insertions are not
perfectly stable (36); and (iii) transposition presents some
site specificity. Some of these drawbacks are absent in shuttle
mutagenesis, where cloned neisserial DNA is mutated by transposition
within E. coli and subsequently transferred into
Neisseria, where it inactivates the corresponding genes via
allelic exchange (33). This technique has been refined with
the construction of versatile minitransposons that permit the creation
of lacZ transcriptional fusions (6), the
production of phoA fusions (5, 12) for the
identification of exported proteins that may play a role in the
pathogenesis (14), and signature tag mutagenesis of N. meningitidis (7). Although it makes possible the
creation of large libraries of mutants, which are predicted to be
stable due to the use of minitransposons, this method is limited in
part by the fact that there are genes refractory to cloning in E. coli which are difficult to mutate.
Because in vitro transposition systems, using mobile elements such as
Tn7 (11), Himar1 (2), and
Ty1 (30), are particularly suitable for the mutagenesis of
naturally competent bacteria, as has been demonstrated in
Haemophilus influenzae and Streptococcus pneumoniae, we reasoned that the use of such a system in N. meningitidis would overcome the problems associated with the
previous transposition systems. Indeed, in addition to the advantages
previously listed for shuttle mutagenesis, in vitro transposition does
not require the target DNA to be cloned. DNA is mutated in vitro, using
a minitransposon and a purified transposase, and is reintroduced into
the bacterium to be mutated, where it inactivates the corresponding genes via allelic exchange. Because Himar1 mariner
transposition is random and very efficiently mediated by a single
protein that is easily overproduced and purified in E. coli
(18), we chose to use this mobile element to develop an in
vitro transposon mutagenesis method for N. meningitidis. We
thus constructed a minitransposon consisting of Himar1
inverted repeat sequences flanking a gene coding for kanamycin
resistance in N. meningitidis and an uptake sequence
(10) that is required for the DNA to be taken up during natural transformation. We demonstrated that this Himar1
derivative efficiently transposed on PCR products, allowing the
site-specific mutagenesis of defined genes, but also on chromosomal
DNA, thereby permitting for the first time the creation of
comprehensive mutant libraries. Therefore, in conjunction with the
genome sequence, in vitro Himar1 transposition allows a
genomic-scale mutational analysis of N. meningitidis.
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MATERIALS AND METHODS |
Bacterial cultures.
E. coli was routinely grown on
liquid or solid Luria-Bertani medium, and kanamycin was used at 20 µg/ml when required.
Because N. meningitidis Z2491, whose genome sequence has
been recently completed by the Sanger Centre (24), is not
transformable, we opted for Z5463, formerly designated C396, another
strain that was isolated from a patient with meningitis in The Gambia
in 1983 (1). Z5463 is a naturally transformable serogroup A
strain that belongs to the same clonal group as Z2491, thus allowing the use of the genomic sequence of the latter strain. N. meningitidis was grown on GCB agar medium (Difco) containing the
Kellog supplements (16), i.e., 4 g of glucose per
liter, 200 ng of thiamine per liter, 5 mg of ferric nitrate per liter,
and 100 ng of L-glutamine per liter. The plates were
incubated for 16 h at 37°C in a moist atmosphere containing 5%
CO2.
To assay maltose catabolism by
N. meningitidis mutants,
individual clones were grown for 6 h at 37°C on 96-well
microtiter
plates containing GCB liquid medium with 2% of a solution
of phenol
red (2 g/liter) and supplemented with the Kellog supplements,
except that glucose was replaced by maltose. Sugar-degrading bacteria
change the color of the medium from red to
yellow.
Molecular biology techniques.
Routine molecular biology
techniques were carried out as recommended (32). DNA
sequences, performed with the Big-Dye primer sequencing kit, were read
on an ABI-Prism 310 automated sequencer (PE Applied Biosystems).
Southern blot analysis was done as previously described
(25).
N. meningitidis chromosomal DNA was prepared as follows from
overnight (ON) cultures on GCB agar plates. A loopful of bacteria
from
one plate was resuspended in 500 µl of lysis buffer (50 mM
NaCl, 20 mM EDTA, 50 mM Tris [pH 8], 1% sodium dodecyl sulfate,
100 µg of
proteinase K), and the sample was incubated for 5 to
10 min at 42°C.
The clear lysate was extracted several times with
phenol-chloroform,
and the DNA was concentrated by ethanol
precipitation.
Construction of vectors.
Plasmid pMM2611 containing the
mini-Himar1, consisting of the first and last 100 bp of
Himar1, was constructed by PCR-ligation-PCR mutagenesis
(3) using pMM26 as a template (19). The primers used for the first half-reaction were T2828r
(5'-TGAAAAAGGAAGAGTATGAG-3') and 76rSma
(5'-TACCCGGGAATCATTTGAAGGTTGGTAC-3'). T2380f
(5'-TTACATGATCCCCCATGTTG-3') and 1218f
(5'-TCGCTCTTGAAGGGAACTATG-3') were used for the second half-reaction. The assembly reaction used the ligation products from
each half-reaction and primers T2828r and T2380f. The final PCR product
was cloned into the EcoRV site of pCDNAII (Invitrogen). The
transposon donor vector pSM1, which was used as a donor for all in
vitro transposition reactions, was constructed by cloning the
aphA-3 gene (41), encoding kanamycin resistance
(Kmr) in Neisseria, and a neisserial uptake
sequence (10) into the mini-Himar1 present on
plasmid pMM2611. The 1.5-kbp kanamycin resistance cassette was
amplified using primers Km6
(5'-CGGGATCCGCCGTCTGAACCAGCGAACCATTTGAGG-3'), where the uptake sequence (10) is in bold, and Km7
(5'-ACGCGTCGACGCTTTTTAGACATCTAAATCTAGG-3'). The PCR fragment
was made blunt ended with T4 DNA polymerase and cloned into the unique
SmaI site of pMM2611.
The
ssa1 gene, previously identified by subtractive
hybridization (
26), was chosen as a target in our
site-specific mutagenesis
experiments. It was PCR amplified from Z5463
chromosomal DNA using
primers oligo R (5'-CGTGCCTGAAATGTCGTTAC-3')
and oligo F (5'-ACCCCAACCTTCCCTACAAA-3').
Where
indicated, the 1.5-kbp PCR product was directly cloned into
the
pCR2.1-TOPO vector using the TOPO TA cloning kit (Invitrogen)
as
outlined by the
manufacturer.
Transformation of N. meningitidis.
An optimized
transformation protocol was designed for this study to detect rare
transposition events. Bacteria grown ON on GCB plates were resuspended
at a final optical density at 550 nm of ca. 5 in transformation buffer,
i.e., supplemented GCB liquid medium containing 5 mM MgCl2.
Aliquots (200 µl) of the previous bacterial suspension were
transferred into the wells of a 24-well tissue culture plate, and
approximately 1 µg DNA was added. Transformation mixtures were
incubated for 30 min at 37°C with strong shaking. Then 1.8 ml of
prewarmed transformation buffer was added, and incubation was continued
for 2 h at 37°C. It is important to note that during this
outgrowth, no increase of the bacterial population was observed.
Transformants were selected on GCB agar plates containing 200 µg of
kanamycin per ml. We reproducibly obtained approximately 8 × 105 transformants/µg of chromosomal DNA.
In vitro transposition reactions.
Purification of a
hyperactive C9 mutant transposase of Himar1 (17)
and in vitro transposition reactions (18) were performed essentially as described previously. However, some parameters known to
enhance the rate of transposition in vitro (19), such as
incubation time, temperature, and MgCl2 concentration, were optimized in our reactions. Transposition reactions were carried out in
10% glycerol-2 mM dithiothreitol-250 µg of bovine serum albumin
per ml-25 mM HEPES [pH 7.9]-100 mM NaCl-10 mM MgCl2.
They contained, in a final volume of 20 µl, at least 500 ng of donor and target DNAs and 120 nM transposase. After a 3-h incubation at
30°C, a 10-min exposure at 75°C was done to inactivate the transposase and the reaction products were directly purified using the
Qiaex II gel extraction kit (Qiagen). Before the DNA was transformed into N. meningitidis, single-stranded gaps, introduced upon
Himar1 transposition (18), were repaired. Gaps in
the DNA were first filled, for 30 min at 16°C, with T4 DNA polymerase
in a final volume of 20 µl. Reactions were carried out in 1× buffer
2 (NEB) containing 50 ng of bovine serum albumin per µl and 1 mM
concentrations of each deoxynucleoside triphosphate. The enzyme was
heat inactivated by a 10-min exposure at 75°C. After addition of 2 µl of ligation mix (22 mM ATP, 5 U of T4 DNA ligase), the mixture was
further incubated ON at 16°C. Usually half of the repaired
transposition products (11 µl) were used to transform N. meningitidis. Clones that had inserted the transposon into the
chromosome were selected on kanamycin-containing plates.
Mapping of Himar1 insertion sites.
Because the
identification of genomic DNA sequences flanking the transposon in
transposon mutants is a long and labor-intensive procedure, especially
when numerous clones are to be analyzed, we used a simple and efficient
ligation-mediated PCR technique (LMPCR) that was previously used for
identifying the flanking sequences in mycobacterial transposon mutants
(29). The linkers were formed by annealing LMP1
(5'-TAGCTTATTCCTCAAGGCACGAGC-3') with LMP2
(5'-GATCGCTCGTGC-3') or LMP3
(5'-CCGGGCTCGTGC-3'); the underlined
sequences correspond to complementary sequences in the primers, whereas
sequences complementary to cohesive ends generated by Sau3AI
(LMP2) or NgoAIV (LMP3) are in bold. Sau3AI and
NgoAIV were chosen because they generate relatively short DNA fragments upon restriction of N. meningitidis
chromosomal DNA. The complete genomic sequence of Z2491 (24)
contains, on average, one Sau3AI site every 1,001 bp and one
NgoAIV site every 1,728 bp. After ligation of the linkers to
digested genomic DNA, the insertion sites were amplified with AmpliTaq
Gold DNA polymerase (PE Applied Biosystems) using LMP1 and IR1
(5'-CCGGGGACTTATCAGCCAACC-3'), an outward primer internal to
the 27-bp inverted repeats of the Himar1 element. PCR
products were gel purified using the Qiaex II gel extraction kit and
directly sequenced using LMP1 as a primer. When the amplified fragments
were too long to be sequenced in extenso, they were cloned using the
TOPO TA cloning kit before being sequenced using suitable primers from
the kit. Sequences were mapped onto the genome of N. meningitidis Z2491 using the BLASTN (4) and Artemis
programs of the Sanger Centre
(http://www.sanger.ac.uk/Projects/N_meningitidis). When
appropriate, the interrupted ORFs were compared with the National
Center for Biotechnology Information database by using the TBLASTN
program (4).
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RESULTS |
Transposition on defined DNA fragments: site-specific
mutagenesis.
As already mentioned, an important aspect of
genomic analysis is the capability to engineer, in the
shortest possible time, numerous site-specific mutations in order to
identify the phenotypes linked with the proteins encoded by defined
genes. To test the feasibility of Himar1 transposition for
this purpose in N. meningitidis, we used as a target
the neisserial hrtA locus, which presents significantly enhanced transformation and recombination efficiencies in
the meningococcus (7), to increase the probability of
detecting transposon mutants. The covalently closed circular (CCC) pHC6 plasmid (7), containing the hrtA locus as a
4.5-kbp EcoRI fragment, was thus subjected in vitro to
Himar1 mutagenesis. Transforming a single
transposition reaction product into N. meningitidis
Z5463 typically gave 2,000 Kmr transformants. A total of 20 randomly picked colonies were analyzed by Southern blotting of
ClaI-digested chromosomal DNA, using the pHC6 plasmid as a
probe. Because there are two ClaI sites 2 kbp apart in the
hrtA locus, three hybridizing fragments of 7.2, 2.2, and 1.8 kbp, respectively, were obtained with wild-type Z5463 DNA (Fig.
1A). Because ClaI does not cut
in the mini-Himar, we expected transposon mutants to present
equally three hybridizing fragments, one of which should present a
1.6-kbp increase in length corresponding to the size of the inserted
mini-Himar1. Indeed, all of the analyzed Kmr
transformants, four of which are shown in Fig. 1A, presented the same
hybridizing pattern, with three ClaI fragments of 7.2, 3.8, and 1.8 kbp. Therefore, the 1.6-kbp increase in length observed for the
central ClaI fragment, 2.2 kbp in the wild type, confirmed that the transformants indeed resulted from the transposition of
Himar1 into the hrtA locus.
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FIG. 1.
Southern blot analysis (A) and mapping of
Himar1 insertions into the hrtA locus (B) of
representative transposon mutants. (A) The target fragment containing
the hrtA locus was either cloned (CCC plasmid) or linear.
Genomic DNA of eight mutants was digested with ClaI and
probed with the pHC6 plasmid containing the hrtA locus on a
4.5-kbp EcoRI fragment. N. meningitidis Z5463
genomic DNA (WT) was included as a control. Molecular sizes are
indicated in kilobase pairs. (B) Distribution of eight insertions on
the ClaI restriction map of the hrtA
genomic locus. The sequences of the insertions sites are
highlighted, and the target dinucleotide that is duplicated upon
transposition is underlined. Note that the mutants whose
Himar1 insertion sites were sequenced are different from the
mutants shown in panel A.
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To test the feasibility of using uncloned DNA as a target for
Himar1 mutagenesis, the
hrtA locus was also
mutagenized as a
gel-purified 4.5-kbp
EcoRI fragment. When a
single mutagenesis
reaction product was transformed into
N. meningitidis Z5463, 200
Km
r transformants were
selected. There was thus a 10-fold decrease
in the number of
transformants compared to the previous experiments,
where CCC DNA was
used as a target. This might reflect the different
conformation of the
DNA in the CCC plasmid, to which
Himar1 is
known to be
sensitive (
19). Southern blot analysis of eight
randomly
picked transformants, four of which are shown in Fig.
1A, confirmed
that the tested clones were transposition mutants.
Indeed, they
presented one hybridizing
ClaI fragment that was
1.6 kbp
longer than the corresponding fragment seen in the wild-type
strain.
Interestingly, unlike what was observed when CCC DNA was
used as the
target, transposon insertions were more evenly scattered
within the
target
hrtA locus: five insertions in the central 2.2-kbp
ClaI fragment, two in the left 7.2-kbp fragment, and one in
the
right 1.8-kbp fragment (Fig.
1A). To easily identify sequences
flanking the
Himar1 after insertional mutagenesis and thus
analyze
the randomness of the transposition, we adapted LMPCR
(
29) to
Neisseria. After amplifying them by
LMPCR, we sequenced and mapped
the
Himar1 insertion sites in
the
hrtA locus for eight mutants
(Fig.
1B). Except for the
insertion sites of mutants 6 and 8,
in the left 7.2-kbp
ClaI
fragment (Fig.
1B), which were identical
and thus probably siblings,
the mapping revealed that the insertion
sites were different,
demonstrating that
Himar1 displayed no apparent
site
specificity. As previously noted by others (
18,
31),
transposition occurred mainly after a TA dinucleotide (in seven
of
eight mutants) that was consequently
duplicated.
To demonstrate the general validity of
Himar1 in vitro
transposition for site-specific mutagenesis in
N. meningitidis, transposition
was performed on a neisserial locus
presenting a normal rate of
transformation, unlike
hrtA. We
chose as a target the
ssa1 gene,
which was previously
identified by RDA subtractive hybridization
(
26). This gene
was particularly interesting because it is absent
from the commensal
N. lactamica and is homologous to a serine
protease, a
virulence-associated protein from
Pseudomonas fluorescens (
26). We amplified by PCR part of the
ssa1 gene
from
N. meningitidis on a 1.5-kbp fragment and subjected it
to
Himar1 in vitro transposition
either directly or after
cloning. As expected, we obtained fewer
Km
r transformants
than in the previous experiment with the highly
transformable
hrtA locus. Typically, we obtained 5 to 10 Km
r
transformants per single mutagenesis reaction. Interestingly,
as
opposed to the previous results with the
hrtA locus, the
prior
cloning of the PCR fragment did not seem to favor
Himar1 transposition.
Several Km
r transformants
were analyzed by Southern blotting, using the
ssa1 PCR
product as a probe (Fig.
2A).
ClaI-digested wild-type Z5463
DNA, included as a control,
gave two hybridizing fragments of
4.8 and 10.8 kbp because the target
contains a
ClaI restriction
site (Fig.
2A). As in the
previous experiments, all the tested
transformants were transposition
mutants because they presented
one hybridizing fragment with a 1.6-kbp
increase in length, corresponding
to the size of the inserted
mini-
Himar1. Of four mutants analyzed,
three had insertions
in the right 4.8-kbp
ClaI fragment whereas
one had the
mini-
Himar1 inserted in the left 10.8-kbp
ClaI
fragment
(Fig.
2A). The insertions sites of
Himar1 in the
different mutants
were amplified by PCR using as primers a sequence in
the
Himar1 inverted repeat and a sequence in the target
gene. The different
lengths of the amplified fragments (data not shown)
confirmed
that the four transposon insertion sites were different,
which
was evidenced by sequencing and mapping them on the
ssa1 genomic
locus (Fig.
2B). In all four mutants,
transposition occurred after
a TA dinucleotide that was consequently
duplicated. In conclusion,
in vitro
Himar1 transposition is
of general use for site-specific
mutagenesis in
N. meningitidis.
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FIG. 2.
Southern blot analysis (A) and mapping of the insertions
into the ssa1 gene (B) of four representative transposon
mutants. (A) Genomic DNA of N. meningitidis Z5463 (WT) and
of four mutants (1 to 4) resulting from transposition into the
ssa1 gene was digested with ClaI and probed with
the 1.5-kbp PCR fragment corresponding to ssa1 that was used
as a target for transposition. Molecular sizes are indicated in
kilobase pairs. (B) Distribution of the insertions on the
ClaI restriction map of the ssa1 genomic
locus. The sequences of the insertion sites are highlighted, and the
target dinucleotide that is duplicated upon transposition is
underlined.
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Transposition on chromosomal DNA: random mutagenesis.
Considering the above results, we expected in vitro Himar1
transposition to be a suitable tool for the creation of comprehensive transposon mutant libraries in N. meningitidis, the lack of
which has been an important obstacle to the genetic characterization of
its pathogenic propensity. We therefore performed Himar1
mutagenesis on Z5463 chromosomal DNA and reproducibly obtained up to
10,000 Kmr colonies per single reaction.
ClaI-digested DNA of more than 50 clones, of which 15 are
shown in Fig. 3A, was analyzed by
Southern blotting using the aphA-3 cassette as a probe. The
hybridization patterns for the tested mutants, singly hybridizing
ClaI fragments of different sizes (Fig. 3A), were in
agreement with a random distribution of Himar1
insertions on the chromosome. This was confirmed by Southern
analysis of EcoRV-digested chromosomal DNA (data not
shown). By using LMPCR (29), we amplified the
Himar1 insertion sites in 45 mutants. The sequencing of the
amplified fragments again confirmed that transposition occurred
at random with no apparent site specificity (Table
1). The positioning of the insertion
sites on the N. meningitidis Z2491 genomic
sequence showed that they were present within every quadrant of
the genome (Fig. 4),
suggesting that there is no obvious hot spot for
Himar1 transposition. The chromosomal locations into which
transposition had occurred were readily identified because our raw
sequences, except for minor sequencing errors, were almost
identical to parts of the complete sequence of Z2491
(24). Because the coding density of Z2491 is 82.9%
(24), we expected a similar percentage of the sequenced
transposition sites to be within ORFs. Indeed, in agreement with a
random distribution of Himar1 in the chromosome, in 35 of 45 mutants (77.8%) transposition occurred within ORFs, 42.9% of which
could not be ascribed a potential function (Table 1).
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FIG. 3.
Southern blot analysis of random insertions into the
chromosome. Genomic DNA of 15 (1 to 15) representative random
transposon mutants (A) and four (mal1 to mal4) mutants that present an
impaired ability to utilize maltose (B) was digested with
ClaI and probed with a PCR product that corresponds to the
kanamycin resistance gene present in the minitransposon. Molecular
sizes are indicated in kilobase pairs.
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FIG. 4.
Distribution of the random Himar1 insertions
on the genome map of N. meningitidis Z2491. The insertions
sites were localized by comparing their sequences (see Table 1) to the
genomic database at the Sanger Centre.
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Another classical possibility to confirm that transposition is random
is to demonstrate that mutants presenting a defined
genetic defect can
be found within the mutant libraries. Therefore,
we decided to
perform a limited screening in search of
N. meningitidis mutants that have lost the ability to utilize
maltose, mainly
for three reasons: (i) maltose utilization can readily
be pinpointed
in vitro by a simple colorimetric test; (ii) this test
has been
used for decades in clinical typing to distinguish between the
meningococcus (Mal
+) and the gonococcus
(Mal
); and (iii) maltose utilization usually depends on
several genes,
which was expected to facilitate the identification of
Mal
mutants in a limited screen such as the one we
intended to perform.
This screening led to the identification of 4 of
480 clones (0.8%
of the tested mutants), denoted mal1 to mal4, with an
impaired
capacity to produce acid during maltose catabolism. Mutants
mal3
and mal4 were clearly Mal
, whereas mal1 and mal2
presented a delayed change of color in
the colorimetric test but were
ultimately Mal
+. The Southern blot analysis revealed that
each mutant presented
a unique hybridization pattern (Fig.
3B),
confirming that they
resulted from different transposition events.
Indeed, the sequences
of the
Himar1 insertion sites in the
four mutants were different
(Table
1). As expected, transposition
occurred within ORFs that
showed similarities to genes encoding
proteins involved in sugar
transport and catabolism (Table
1). In mal1,
Himar1 was inserted
within a gene encoding a putative
respiratory
D-lactate dehydrogenase,
a membrane-bound
flavoenzyme that feeds electrons into the respiratory
chain in
E. coli (
22). Therefore, the delayed Mal
+
phenotype in the mal1 mutant suggests that membrane potential
is
important in the meningococcus for maltose import or degradation.
In
mal2, mal3, and mal4, mini-
Himar1 insertions occurred within
a 6-kbp region (Fig.
5). The mal2 and
mal3 mutants presented different
insertions into the same ORF, encoding
a maltose phosphorylase,
an enzyme catalyzing the phosphorolysis of
maltose into glucose-1-phosphate.
In
E. coli
(
22), phosphoglucomutase converts glucose-1-phosphate
into
glucose-6-phosphate, which subsequently enters the glycolysis
pathway.
Interestingly, an ORF homologous to the
-phosphoglucomutase
gene
from
Lactococcus lactis was found after the maltose
phosphorylase
gene and is probably cotranscribed with it, because the
two ORFs
are separated by only 12 bp (Fig.
5). However, a polar effect
of the inserted transposon on the phosphoglucomutase gene can
be
excluded because this gene is duplicated elsewhere in the genome.
The
Mal
phenotype of mal3 suggests that the maltose
phosphorylase is
essential for the entry of maltose into the glycolysis
pathway.
On the other hand, the Mal
+ phenotype of mal2 is
probably explained by the fact that
Himar1 transposed near
the 3' end of the gene without entirely disrupting
its function (Fig.
5). Mal4 was mutated in an ORF with similarities
to a gene from carrot
encoding a sucrose/H
+ symporter. The Mal
phenotype of mal4 therefore suggests that the interrupted gene
may
encode a maltose/H
+ symporter that could be responsible for
the entry of maltose
into the cell. However, because the interrupted
ORF is probably
cotranscribed (Fig.
5) with an ORF that is present 3 bp
downstream,
homologous to a gene encoding mutarotase from
Streptococcus thermophilus catalyzing the interconversion of
the
and
anomeric forms of
sugars, a polar effect, though
unlikely, cannot be ruled out.
View larger version (13K):
[in this window]
[in a new window]
|
FIG. 5.
Himar1 insertions in mutants mal2, mal3, and
mal4 occur within a 6-kbp region of the N. meningitidis
genome. The ORFs, numbered as in the Z2491 sequence (24),
encode a -phosphoglucomutase (Nm2097), a maltose phosphorylase
(Nm2098), a mutarotase (Nm2099), and a putative maltose/H+
symporter (Nm2100).
|
|
| |
DISCUSSION |
We have developed an extremely simple transposon-mediated
mutagenesis system, based on in vitro Himar1 transposition
(18), that is suitable for mutagenesis of N. meningitidis both in a site-directed (defined genomic DNA
fragments) and in a random (chromosomal DNA) fashion. As opposed to
previously available mutagenesis methods, in vitro Himar1
transposition presents several particularly desirable features that
make it suitable for a genomic-scale mutagenesis of the
meningococcus. The mobile element used for mutagenesis is a small
minitransposon, approximately 1.6 kbp, that consists only of a
kanamycin resistance gene and a neisserial uptake sequence
(10) flanked by the inverted repeats of Himar1. Because the transposon lacks the gene encoding the transposase, it can
no longer transpose once it is inserted in the chromosome, which is
expected to render the mutants perfectly stable. The small size of the
mini-Himar1 and the presence of convenient restriction sites will facilitate the possible construction of minitransposons containing either promoterless reporter genes, such as
lacZ, phoA, or gfp, or unique short
DNA tags. The construction of tagged transposons opens the way for the
development of signature-tagged mutagenesis (13), a powerful
approach to the identification of virulence genes in vivo. As
demonstrated initially in Salmonella enterica serovar
Typhimurium (13) and subsequently in numerous
pathogenic bacteria (27), signature-tagged mutagenesis could
greatly facilitate the screening of mutant libraries in search of
N. meningitidis mutants affected in virulence.
We demonstrate that neisserial DNA, whether chromosomal or PCR
amplified, is a suitable target for in vitro Himar1
transposition. Therefore, as opposed to allelic exchange and shuttle
mutagenesis, there is no need to clone the target DNA before subjecting
it to mutagenesis. Consequently, genes that are refractory to cloning in E. coli can nevertheless be mutagenized. Moreover, the
use of large PCR fragments, as demonstrated in H. influenzae
(18), could allow the simultaneous site-directed mutagenesis
of many genes. This could facilitate the mutational analysis of large regions of the chromosome that carry numerous genes which are possibly
important for virulence, as, for example, the chromosomal regions found
only in the pathogenic Neisseria species that were identified using subtractive hybridization (26). When
chromosomal DNA was used as a target, the frequency of transposition
was high compared to that for previously available mutagenesis methods, which permitted the creation of mutant libraries of strain Z5463 numbering tens of thousands of mutants. Because N. meningitidis Z2491, which has been sequenced by the Sanger Centre
(24), is not transformable, the very closely related strain
Z5463 seems a good choice for starting a mutational analysis of
N. meningitidis virulence. However, if needed, other
N. meningitidis strains can be mutagenized provided that
they are transformable, keeping in mind that the number of mutants that
can be obtained will be highly dependent on the transformation
efficiency. For example, when in vitro Himar1 transposition
was performed on a variant of 8013, a serogroup C strain
(20) that is less transformable than Z5463, a 20-fold
reduction in the number of mutants obtained was observed (data not shown).
Most importantly, from the sequences of 61 insertion sites
8 in
hrtA, 4 in ssa1, and 49 random insertionsit can
be deduced that Himar1 transposition occurs with no apparent
site specificity. All the mutants we tested resulted from independent
transposition events, except two mutants with mutations in the
hrtA locus that showed the same point of insertion. In this
case, it is likely that the two clones were siblings even though it
cannot be excluded that this insertion site, nucleotide 290 in the
4,472-bp hrtA locus, is a "warm spot" for
Himar1 transposition. As previously reported by others
(18, 19, 31), in 95% of the analyzed mutants transposition
occurred after TA dinucleotides. This very limited preference is in no
way a problem for N. meningitidis random mutagenesis, since
TA dinucleotides occur, on average, approximately every 17 bp in the
genome. However, even the specificity for TA dinucleotides could be
relaxed by replacing Mg2+ with Mn2+ in the
transposition reaction mixtures (18). Another demonstration that Himar1 transposition was random came from a limited
screen in search of mutants presenting an impaired maltose catabolism. Of the analyzed mutants, 0.8% presented an impaired capacity to produce acid from maltose degradation and were mutated within genes
that showed similarities to those encoding proteins involved in sugar
transport and catabolism. Our results suggest that maltose import into
N. meningitidis may be driven by the flow of protons across
the membrane, an unorthodox uptake system for maltose already described
in Lactobacillus sanfranciscensis (23).
Interestingly, this was also suggested by the recent in silico analysis
of the complete sequence of MC58 (38). However,
comprehensive mutational and biochemical analyses are of course
required to give a broader view of the maltose catabolic pathway in the
meningococcus. Moreover, our results allow us to speculate on the
reasons why N. gonorrhoeae is naturally Mal.
Indeed, a search of the preliminary genome sequence of the gonococcus at the University of Oklahoma (http://dna1.chem.ou.edu/gono.html) revealed that the maltose phosphorylase and the putative
maltose/H+ symporter genes, the genes we identified
as specific to maltose catabolism, are both present in N. gonorrhoeae as pseudo-genes. They both contain frameshifts and/or
stop codons. Hence, the inability of N. gonorrhoeae to
utilize maltose is the result of an evolutionary process during which
the gonococcus accumulated mutations in genes of the maltose catabolic
pathway and ultimately lost the capacity to utilize maltose.
In conclusion, in vitro Himar1 transposition should allow
N. meningitidis mutational analysis on a scale that was
previously unfeasible. Hopefully, mutagenesis studies, in conjunction
with the genome sequences (24, 38), will lead to a better
understanding of the molecular mechanisms of meningococcal meningitis,
a disease that remains an important public health burden
(40).
| |
ACKNOWLEDGMENTS |
We thank U. Vogel and M. Frosch (University of Würzburg)
for providing plasmid pHC6. We thank J.-L. Beretti for help in
purifying the Himar1 transposase. We are grateful to C. R. Tinsley and J.-M. Reyrat for critical reading of the
manuscript. E. Abachin and G. Quesne are acknowledged for
automated sequencing. The Sanger Centre and the Gonococcal Genome
Sequencing Project (University of Oklahoma) are gratefully acknowledged
for making genome sequences publicly available before publication.
This work was supported by grants from the Institut National de la
Santé et de la Recherche Médicale, Université Paris V-René Descartes, and Fondation pour la Recherche Médicale.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: INSERM U411,
Laboratoire de Microbiologie, Faculté de Médecine
Necker-Enfants Malades, 156, rue de Vaugirard, 75015 Paris, France.
Phone: 33-140615483. Fax: 33-140615592. E-mail:
pelicic{at}necker.fr.
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[Abstract]
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Abstract
Introduction
