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Journal of Bacteriology, September 1998, p. 4750-4752, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Gene Transfer between Related Bacteria by Electrotransformation:
Mapping Salmonella typhi Genes in Salmonella
typhimurium
Cecilia S.
Toro,1
Guido C.
Mora,1 and
Nara
Figueroa-Bossi2,*
Departamento de Genética Molecular y
Microbiología, Facultad de Ciencias Biologicas, Pontificia
Universidad Católica de Chile,1 and
Centre de Génétique Moléculaire du CNRS,
91198 Gif-sur-Yvette Cedex, France2
Received 17 April 1998/Accepted 6 July 1998
 |
ABSTRACT |
Transfer of newly isolated mutations into a fresh background is an
essential step of genetic analysis and strain construction. Gene
transfer is hampered in Salmonella typhi and in other
pathogenic bacteria by the lack of a generalized transduction system.
We show here that this problem can be partially circumvented by using electrotransformation as a means for delivering S. typhi
DNA into suitable S. typhi or Salmonella
typhimurium recipients. Transferred DNA can recombine with the
homologous region in the host chromosome. In one application of the
method, mutations isolated in S. typhi were genetically
mapped in S. typhimurium.
| |
TEXT |
Typhoid fever is a complex systemic
infection quite widespread in developing countries (17).
Since its causative agent, Salmonella typhi, is a strictly
human pathogen, it has been difficult to find an appropriate animal
model for virulence studies. This limitation is partially overcome by
the use of human cell lines for in vitro studies. Similar to the
results obtained for other bacterial pathogens, this approach is
beginning to yield information on the genes involved in invasion and
other virulence determinants (3, 4, 7).
Analysis of the S. typhi chromosome has revealed some
major differences with respect to the closely related bacteria
Salmonella typhimurium and Escherichia coli. Most
notable is the inversion of large chromosomal segments thought to
result from recombination between rRNA loci (11-13). In
spite of these differences, the gene order within the inverted regions
and elsewhere in the chromosome is virtually the same in S. typhi as in S. typhimurium and the two bacterial
serovars share more than 90% homology at the DNA sequence level
(5). Indeed, segments of the S. typhimurium chromosome can undergo recombination with the homologous region in the
S. typhi chromosome once the natural barrier imposed by the mismatch repair system is eliminated by mutation (mutS)
in the recipient strains (22, 23).
A major problem encountered in the genetic analysis of S. typhi is the lack of a convenient gene transfer system and, in
particular, of a generalized transducing phage comparable to phage P22
of S. typhimurium. While P22 will deliver DNA into
S. typhi, making S. typhimurium-S.
typhi crosses possible (22, 23), it is incapable of
multiplying inside this host, thus preventing gene transfer in the
opposite direction, i.e., from S. typhi to
S. typhimurium, or between S. typhi
strains. To try to circumvent this problem, we sought to test whether
S. typhi genetic material introduced into S. typhimurium cells by electrotransformation could undergo homologous recombination.
Transformation of S. typhimurium with linear
DNA.
As a preliminary test of the method we used a cloned DNA
fragment from S. typhimurium as input material. The DNA
insert of plasmid pCV47 contains the entire S. typhimurium leucine operon plus approximately 13 kb of
neighboring DNA (20). This insert is released by
BamHI treatment as an 18.5-kb DNA fragment. pCV47 DNA,
either cleaved with BamHI or untreated, was used to
transform S. typhimurium strains in which the
leuA gene was inactivated by a MudJ insertion. Three
recipient strains were compared: MA2290, which expresses a functional
RecBCD enzyme; MA5133, in which the nuclease activity of RecBCD (Exo V)
is inactivated (recD::Tn10dTc); and
MA5031, which harbors a recBD deletion but is recombination proficient due to an sbc mutation (sbcE21
[8]). Results shown in Table
1 confirm that, as suggested from
previous work with E. coli (18), inactivation of
Exo V in S. typhimurium greatly improves the recovery
of transformants with linear DNA. Leu+ transformants were
found to be kanamycin sensitive and Lac
, consistent with
their resulting from recombination events which replace the
leuA::MudJ insertion with the wild-type
leuA gene. The higher transformation efficiency in the
recBD sbcE background is indicative of the "hyper-rec"
phenotype of this strain (8, 9).
We then evaluated the ability of
S. typhimurium to
yield Leu
+ recombinants when transformed with bulk
chromosomal DNA prepared
from
S. typhimurium or
S. typhi. Results in Table
2 show that
this is indeed possible
provided the nuclease activity of RecBCD
of the recipient strain is
inactivated. Again, the frequency of
recombination is higher in the
sbcE21 background. In addition,
in the experiments involving
S. typhi DNA, the formation of Leu
+
recombinants also requires that the recipient strain be defective
in
mismatch repair (
mutS). This is in agreement with the known
inhibitory effect of the mismatch repair system on recombination
between closely related sequences (
22,
23).
In a separate experiment, chromosomal DNA from an
S. typhi strain carrying a
leuA::MudJ insertion
(constructed by P22 transduction
[
22]) was used to
transform an
S. typhimurium recipient
containing
an intact
leu operon region (MA5100).
Kanamycin-resistant (Kan
r) recombinants were selected.
Although such isolates occurred
at a somewhat lower frequency than the
Leu
+ recombinants described above, the effect of
recD and
mutS was
nearly identical to the data in
Table
2 (data not shown). Southern
analysis confirmed that the
structure of the
leu operon region
in four
independent Kan
r transformants was indistinguishable
from that of donor DNA (data
not shown). Thus, no unusual
rearrangements accompanied the acquisition
of the MudJ insertion by the
recipient strain.
Although the
mutS mutation makes genetic exchanges between
S. typhi and
S. typhimurium possible,
the frequencies with which
recombinants were recovered in the above
experiment are low. We
sought to see whether the efficiency of the
process could be increased
by improving the transformation step.
S. typhimurium SL4213 (
metA22 metE551 galE496
rpsL120 xyl-104 
[Fels2] H1-b
H2-
e,
n,
x nml hsdL6 hsdSA29) was
previously recognized to be a particularly suitable
transformation
recipient owing to
galE and
hsd mutations that
favor DNA uptake and lower the restriction barrier, respectively
(
16). Upon repeating the above experiments with the
SL4213 background,
we observed a 10-fold increase in the efficiency of
recovery of
recombinant clones provided that
recD and
mutS mutations were
both present (strain MA5383, see Table
3, footnote
a). Such an
improvement was not specific to the
leu operon region but was
also observed in exchanges
involving the
his operon and the
proU operon (data not shown). Unfortunately, limitations in the
availability
of selectable markers hampered the construction of the
triply
mutated SL4213 derivative carrying the
recBD deletion
sbcE21 and
mutS::Tn
10dCm. We
therefore adopted strain MA5383 (Table
3)
for
the mapping experiments described below.
Mapping of S. typhi mutations in S. typhimurium.
The possibility of moving mutations isolated in
S. typhi to S. typhimurium for
mapping analysis was tested with eight independent MudJ insertion
mutants. Five such insertions cause amino acid auxotrophies (Met, Arg,
Ilv, Leu, Phe) and the remaining three confer chlorate resistance. As
schematized in Fig. 1, chromosomal DNA
was prepared from each of the eight S. typhi strains
and used to transform strain MA5383. Kanr transformants
were picked, purified, and used as donors in P22-mediated transductional crosses with wild-type S. typhimurium
LT2 as the recipient. Once in the wild-type background, MudJ-associated
mutations were mapped by using the "locked-in" Mud-P22 hybrid
procedure (21). The auxotrophic mutants allowed the direct
selection of prototrophic transductants (Fig. 1). With the
chlorate-resistant insertions, a Tn10dTc element was
introduced within the lac sequence of MudJ and selection was
for the loss of the Tet resistance phenotype (2). The
Mud-P22 lysates that scored positive in the transductional screening
allowed the positioning of the different MudJ insertions in the
S. typhimurium chromosome (Table 3). From these data, the identities of affected loci could be deduced unambiguously for
those resulting in an auxotrophy and preliminarily for the others. In
the case of the MudJ insertion of strain DD46, the initial
identification was recently confirmed by DNA sequence analysis (data
not shown). The expected positions of the various loci in the
S. typhi chromosome were obtained upon correcting for
the known discontinuities between the S. typhimurium
and S. typhi physical maps (11).
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FIG. 1.
Outline of mapping procedure. Step 1. Chromosomal DNA
from an S. typhi strain carrying the MudJ insertion to
be mapped (for example, the argA locus) is extracted and
mildly sheared. Step 2. DNA is used to electrotransform an
S. typhimurium recipient in which the recD
and mutS genes are inactivated. Selection for
Kanr yields recombinants in which the argA locus
on the recipient chromosome has been replaced by the
argA::MudJ marker. (Although the chromosomal
region containing argA is inverted in S. typhimurium relative to S. typhi, the gene order
within this region is conserved between the two bacteria.) Step 3. Recombinant bacteria are spread on a minimal plate, and phage P22
transducing lysates individually enriched for various portions of the
S. typhimurium chromosome are spotted on the bacterial
lawn. Step 4. The lysate enriched for the region carrying the wild-type
allele of the MudJ-disrupted locus gives rise to prototrophic
transductants (patch of colonies in the spotted area).
|
|
In conclusion, the data presented here show that electrotransformation
techniques combined with the use of appropriate host
strains can
partially circumvent the problem resulting from the
lack of a suitable
transduction system in
S. typhi. Although here
we used
genetic mapping as a test of the method, this is not its
only
possible application. In a separate line of work, we successfully
used
this method for moving the MudJ insertions which confer chlorate
resistance (affecting bacterial replication in epithelial cells)
from
the mutagenized background in which they were originally
isolated into
a "fresh"
S. typhi background (
3). Such
backcrosses
were crucial for unambiguously correlating individual
mutations
with their respective phenotypes. Surprisingly, in
these
S. typhi-S. typhi exchanges,
recD mutational inactivation was no longer a
prerequisite
for the recovery of transformants. Similar findings
were recently made
with
E. coli and were ascribed to a transient
inhibition of
Exo V following electroshock conditions (
6).
| |
ACKNOWLEDGMENTS |
We are indebted to Nello Bossi for discussions and comments on the
manuscript and J. R. Roth for strain SL4213 and strain TT16808,
which carries the mutS171::Tn10dCm allele used
here. We also thank reviewers for constructive criticism.
This work was supported by the ECOS-CONICYT France-Chile cooperation
program (grant no. C94B06), by FONDECYT grants 1960255 and 2970029, and
by the Centre National de la Recherche Scientifique.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Centre de
Génétique Moléculaire du CNRS, 91198 Gif-sur-Yvette
Cédex, France. Phone: 33-1-69-82-31-79. Fax: 33-1-69 82 32 30;
E-mail: figueroa{at}cgm.cnrs-gif.fr.
| |
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Journal of Bacteriology, September 1998, p. 4750-4752, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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