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Journal of Bacteriology, December 1999, p. 7449-7456, Vol. 181, No. 24
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Gene Disruption through Homologous Recombination in
Spiroplasma citri: an scm1-Disrupted Motility
Mutant Is Pathogenic
Sybille
Duret,
Jean-Luc
Danet,
Monique
Garnier, and
Joël
Renaudin*
Laboratoire de Biologie Cellulaire et
Moléculaire, INRA et Université Victor Segalen Bordeaux
2, 33883 Villenave d'Ornon Cedex, France
Received 10 June 1999/Accepted 27 September 1999
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ABSTRACT |
To determine whether homologous recombination could be used to
inactivate selected genes in Spiroplasma citri, plasmid
constructs were designed to disrupt the motility gene scm1.
An internal scm1 gene fragment was inserted into plasmid
pKT1, which replicates in Escherichia coli but not in
S. citri, and into the S. citri oriC plasmid
pBOT1, which replicates in spiroplasma cells as well as in E. coli. Electrotransformation of S. citri with the
nonreplicative, recombinant plasmid pKTM1 yielded no transformants. In
contrast, spiroplasmal transformants were obtained with the
replicative, pBOT1-derived plasmid pCJ32. During passaging of the
transformants, the plasmid was found to integrate into the chromosome
by homologous recombination either at the oriC region or at
the scm1 gene. In the latter case, plasmid integration by a
single crossover between the scm1 gene fragment carried by
the plasmid and the full-length scm1 gene carried by the
chromosome led to a nonmotile phenotype. Transmission of the
scm1-disrupted mutant to periwinkle (Catharanthus roseus) plants through injection into the leafhopper vector
(Circulifer haematoceps) showed that the motility mutant
multiplied in the insects and was efficiently transmitted to plants, in
which it induced symptoms similarly to the wild-type S. citri strain. These results suggest that the spiroplasmal
motility may not be essential for pathogenicity and that, more broadly,
the S. citri oriC plasmids can be considered promising
tools for specific gene disruption by promoting homologous
recombination in S. citri, a mollicute which probably lacks
a functional RecA protein.
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INTRODUCTION |
Spiroplasmas are eubacteria
belonging to the class Mollicutes, a group of wall-less
organisms which represents a branch in the phylogenetic tree of the
gram-positive bacteria (41). In addition to the lack of cell
wall, mollicutes are characterized by small genome size, low number of
rRNA operons and tRNA genes, and limited metabolic activities. The
molecular features of mollicutes have been extensively reviewed
(1, 12, 31). Among mollicutes, spiroplasmas are
characterized by helical morphology and motility (7-9, 21).
Spiroplasmas are associated primarily with arthropods, mainly insects,
and three of them, Spiroplasma citri, Spiroplasma kunkelii, and Spiroplasma phoeniceum, are pathogenic to
plants (44). In plants, the organisms are located in, and
restricted to, the phloem sieve tubes. They are transmitted from plant
to plant by sap-feeding leafhopper vectors. S. citri was the
first plant-pathogenic mollicute to have been cultured and
characterized as the etiological agent of citrus stubborn disease
(34). However, in spite of extensive characterization of
S. citri, very little is known about the genetic
determinants that govern the interactions between the spiroplasma and
the two hosts in which it multiplies, the insect vector and the plant
(2, 3, 13). In recent years, molecular tools have been
developed for genetic analysis of S. citri. Plasmid vectors
were constructed by combining the chromosomal replication origin of
S. citri (oriC region) and the tetM
gene of Tn916 to express cloned genes in S. citri
(32, 33, 45). Also, transposon (Tn4001)
mutagenesis was used to produce S. citri mutants affected in
motility or in plant pathogenicity, and functional complementation of
these mutants was achieved by transformation with oriC
plasmids carrying the wild-type DNA (15-17, 19). However,
in S. citri, specific gene disruption through homologous
recombination has not yet been obtained.
In mollicutes, homologous recombination was first demonstrated to occur
in Acholeplasma oculi (24, 25), then in
Acholeplasma laidlawii (11) and Mycoplasma
gallisepticum (6), and very recently in
Mycoplasma genitalium (10). In previous studies (26, 27), we have shown that S. citri probably
lacks a functional recA gene, suggesting that this organism
is unable to undergo generalized homologous recombination dependent on
the RecA protein. However, we have also shown that despite the absence
of RecA protein, homologous recombination does occur in specific cases
(26, 33). In particular, when S. citri ASP1 was
transformed with the oriC plasmid pBOT1, it was shown that
during passaging of spiroplasmal transformants, the plasmid integrated
into the host chromosome by homologous recombination at the
oriC region (33).
In the present study, we used a replicative, pBOT1-derived
oriC plasmid to inactivate the S. citri motility
gene scm1 through homologous recombination. The
scm1 gene codes for a putative highly hydrophobic protein of
409 amino acids (19). This polypeptide was predicted to
contain an N terminus characteristic of signal peptide sequences as
well as 10 transmembrane 
helices and a leucine zipper-like motif.
However, the Scm1 protein does not show homology with known proteins,
and its function is unknown. We chose to inactivate the motility gene
scm1 for the following reasons: (i) the nucleotide sequence
of the scm1 gene has been determined, (ii) it has been shown
that disruption of scm1 by transposon insertion gives rise
to a readily observable, nonmotile phenotype, and (iii) inactivation of
the scm1 gene has no significant effect on the spiroplasma
growth rate (19). In these early studies, an insertional
mutant, G540, was obtained by transformation of a high-passage culture
of S. citri GII-3 which had lost its ability to be
transmitted by the leafhopper vector. In the present work, the
isolation of a motility mutant by disruption of the scm1
gene in a low-passage, transmissible isolate of S. citri
GII-3 was made to investigate the putative role of motility in the
pathogenicity of S. citri. The scm1-disrupted
mutant was experimentally transmitted to periwinkle plants through
injection to the leafhopper vector, and it was found to be pathogenic.
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
E. coli TG1
{supE hsd
5 thi (lac-proAB)
F'[traD36 proAB+
lacIq lacZM15]}, an
EcoK
derivative of JM101, was used as the host strain for
subcloning experiments and for propagation of plasmids pBOT1, pCJ3,
pKT1, pKTM1, and pCJ32. Plasmids pBOT1 and pCJ3, carrying the S. citri oriC region and the scm1 gene, respectively, have
been described elsewhere (19, 32, 33). Plasmid pKT1 was
obtained by inserting the tetM gene, rescued from the pBOT1
plasmid as a 4.2-kbp BamHI-EcoRI fragment, into
the pBluescript II KS+ vector (Stratagene Cloning Systems,
La Jolla, Calif.). Constructions of plasmids pKTM1 and pCJ32 are
described in Results. For transformation with plasmid DNA, E. coli competent cells were prepared according to the procedure
described by Hanahan (18). S. citri GII-3 was originally isolated from its leafhopper vector, Circulifer
haematoceps, captured in Morocco (40). From an early
passage of this isolate, a triply cloned strain (38) was
obtained and used in this study. This cloned strain was shown to be
phytopathogenic by transmission to periwinkle (Catharanthus
roseus) through injection to the leafhopper vector C. haematoceps. Spiroplasmas were grown at 32°C in SP4 medium
(42) from which fresh yeast extract was omitted.
Transformation of S. citri.
Electrotransformation of
S. citri with plasmid DNA was done as previously described
(32, 36), with 1 to 30 µg of DNA in the case of the
suicide plasmid pKTM1 and 1 to 5 µg in the case of the replicative
plasmid pCJ32. The S. citri transformants were selected by
plating on solid SP4 medium supplemented with 2 µg of tetracycline
per ml. Individual colonies were picked and grown in broth medium
containing 2 µg of tetracycline per ml. During propagation, the
antibiotic concentration was progressively increased up to 15 µg/ml.
DNA isolation and Southern blot hybridization.
Large-scale
and small-scale preparations of plasmid DNA amplified in E. coli were carried out according to standard procedures (35). Large-scale preparation of spiroplasmal genomic DNA
has been described elsewhere (43). For small-scale
preparation, spiroplasma cells from a 10-ml culture were collected by
centrifugation and resuspended in 1.8 ml of STE buffer (100 mM NaCl, 10 mM Tris-HCl [pH 8], 1 mM EDTA). Cells were lysed by adding 200 µl
of 10% sodium dodecyl sulfate. The lysate was heated at 65°C for 15 min and then treated with 100 µg of RNase for 30 min at 37°C. The
DNA was further purified by phenol-chloroform deproteinization and ethanol-acetate precipitation and finally resuspended in 200 µl of
sterile water or TE buffer (10 mM Tris-HCl [pH 8], 1 mM EDTA). For
Southern blot hybridization, restricted DNA was blotted to positively
charged nylon membranes by the alkali transfer procedure and hybridized
with the 32P-labeled probe under standard stringent
conditions (35).
Primers and PCR amplification.
Primers CJ5
(5'-AATGACGGATCATCAACGG-3'), CJ6
(5'-CAATTACCAACCATGTTAGC-3'), CJ8
(5'-GGTTAGTAATGCTGATCGC-3'), CJ17
(5'-CTTTACAGGGAGATAGTGC-3'), CJ26
(5'-ATTGCTGGGGCAGTTGTTC-3'), and CJ27
(5'-CTAAATATTGTTCACATTAAAGTTTGTC-3') correspond,
respectively, to nucleotides 3928 to 3946, 3576 to 3595, 2328 to 2346, 2678 to 2696, 2572 to 2591, and 3604 to 3632 of the previously
published sequence (19). Primers IS1
(5'-ATATTCTGTAAAGGATGACG-3') and IS3
(5'-CTTTAACAGCTTCTCTG-3') correspond, respectively, to nucleotides 155 to 174, and 923 to 939 of the IS256
nucleotide sequence (5). Primers Tet1
(5'-CTGCAAAAGATGGCGTAC-3') and Tet2 (5'-CGTAAATGTAGTACTCCAC-3') correspond, respectively, to
nucleotides 521 to 538 and 1037 to 1055 of the tetM gene
(4). Primer EV7 (5'-CAATAAGCAAGCATCTGTAATTAG-3')
corresponds to nucleotides 507 to 530 of the nucleotide sequence
of the S. citri oriC region (45). Amplification
was carried out in a 50-µl reaction mixture containing 5 to 20 ng of
target DNA, 50 mM Tris-HCl (pH 8.8), 2 mM MgCl2, 200 µg
of bovine serum albumin per ml, 0.05% W1 detergent, 0.2 mM
deoxynucleoside triphosphates, 1 µM each primer, and 2.5 U of
Taq DNA polymerase (GIBCO/BRL Life Technologies, Inc.,
Gaithersburg, Md.). The reaction was performed in a thermal cycler
(Perkin-Elmer Cetus Corp., Norwalk, Conn.). Amplification was achieved
in over 35 cycles, each of 45 s at 92°C, 45 s at the
annealing temperature, and 1 min at 72°C, with an additional step of
10 min at 72°C. The annealing temperature was set to 52°C for
primer pairs IS1-IS3, Tet1-Tet2, and Reverse-CJ5, 56°C for primer
pairs CJ6-CJ8 and CJ8-EV7, and 58°C for primer pair CJ26-EV7. PCR
products Reverse-CJ5 and CJ26-EV7 were cloned in E. coli by
using the pGEM-T Easy vector (Promega Corporation, Madison, Wis.) and
sequenced with a T7 sequencing kit (Pharmacia Biotech, Uppsala, Sweden).
Experimental transmission assay.
Microinjection of S. citri cultures into C. haematoceps leafhoppers and
transmission to periwinkle (Catharanthus roseus) plants were
carried out as previously described (14, 16). The insects were microinjected with about 0.2 µl of a 108-CFU/ml
S. citri culture, i.e., approximately 2 × 104 spiroplasma cells per insect. For each transmission
assay, 50 injected leafhoppers were caged on five separate young
periwinkle plants (10 insects per plant) for a 2-week period. At the
end of the transmission period, the insects were killed and the plants were kept at 30°C in the greenhouse. Under these conditions, the wild-type strain GII-3 produces severe symptoms within 2 weeks after
the transmission period. Culture of S. citri from plants and
insects as well as transmission through Parafilm membranes have also
been described previously (14, 16). For determination of CFU
per insect, groups of two to five leafhoppers were ground in 2 ml of
SP4 medium. The extracts were passed through filter membranes (pore
diameter, 0.45 µm), and 10-fold serial dilutions were plated. The
number of CFU was determined as the average value of three independent
extracts. In the case of plants, the extracts were prepared from 0.1 to
0.3 g of midribs that were minced with a razor blade in 2 ml of
SP4 medium.
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RESULTS |
Construction of plasmids pKTM1 and pCJ32.
To demonstrate gene
disruption by homologous recombination, an internal fragment of the
scm1 gene was inserted into either a replicative or a
nonreplicative plasmid vector and introduced into S. citri
by electroporation. The scm1 gene fragment was obtained by
PCR amplification of pCJ3 DNA with primers CJ6 and CJ17. The 918-bp
amplification product was initially cloned in E. coli by using the pGEM-T Easy vector system and was then rescued from the
recombinant plasmid either as an EcoRI fragment of 937 bp or
as a Sau3AI fragment of 2,203 bp. The nonreplicative plasmid pKTM1 was obtained by inserting the EcoRI fragment
containing the scm1 gene fragment into the
EcoRI-linearized plasmid pKT1. The replicative plasmid pCJ32
was obtained by inserting the Sau3AI fragment at the
BglII site of plasmid pBOT1, i.e., within the dnaA gene of the oriC fragment. As a result, the
1.95-kbp oriC region was divided into two separated
fragments of 0.6 and 1.35 kbp that we named ori 1 and ori 2, respectively (see Fig. 2). In previous studies (33) we have
shown that the presence of a functional dnaA gene on the
plasmid is not required for plasmid replication and that during
passaging of the spiroplasmal transformants, the pBOT1 plasmid
integrates into the chromosome by homologous recombination at the
oriC region. Hence, insertion of the scm1 gene
fragment into the oriC region of pBOT1 was expected not to prevent plasmid replication and, in addition, to decrease the frequency
of recombination in this region.
Transformation of S. citri with plasmids pKTM1 and
pCJ32.
Electrotransformation of S. citri GII-3 with
different amounts (1 to 30 µg) of the suicide plasmid pKTM1
repeatedly yielded no tetracycline-resistant transformants, indicating
that in these experiments, integration of the plasmid into the host
chromosome by a single crossover did not occur or occurred with a very
low frequency, less than 109 transformants/CFU.
Therefore, to enhance the opportunity for recombination between the
plasmid and the chromosome, the scm1 gene fragment was
introduced into spiroplasma cells by transformation with the
replicative plasmid pCJ32. Transformation with this plasmid yielded
spiroplasmal transformants with a transformation efficiency of 5 × 103 to 104 transformants/µg of plasmid
DNA, corresponding to a frequency of 5 × 106 to
105 transformants/CFU. All of these
tetracycline-resistant colonies showed a motile phenotype similar to
that of the cells transformed with the scm1-free pBOT1 vector.
Behavior of plasmid pCJ32 during propagation of spiroplasmal
transformants.
Thirty-one spiroplasmal transformants were grown in
liquid SP4 medium containing tetracycline, and the presence of the
pCJ32 plasmid in these transformants was demonstrated by PCR
amplification with primer pair Tet1-Tet2 and Southern blot
hybridization of total DNA with the pCJ32 probe (data not shown). To
determine whether the plasmid was maintained extrachromosomally as a
free plasmid or could be integrated into the spiroplasmal chromosome, the spiroplasmal transformants carrying pCJ32 were subcultured for 15 successive propagations in liquid medium, corresponding to
approximately 50 generations. During in vitro propagations, the
behavior of the plasmid was monitored by Southern blot hybridization of
genomic DNA at passages 5, 10, and 15. At passage 5, all 31 spiroplasmal clones tested were found to contain pCJ32 as a free, extrachromosomal DNA. In the experiment represented in Fig.
1A, total DNA from spiroplasmal
transformants was restricted by EcoRI and hybridized with
the pCJ32 probe. All three EcoRI fragments of the purified
pCJ32, with sizes of 1, 4, and 6.5 kbp (lane 1), were detected in the
spiroplasmal transformants (clones 3, 11, and 30 [lanes 3, 4, and 5, respectively]). Two additional fragments larger than 13 and 9 kbp were
faintly detected. These two fragments were identical to those in the
wild-type strain (lane 2), which were previously shown to carry the
scm1 gene and the oriC region, respectively
(19). The detection of these two fragments suggested that no
DNA insertion or recombination had occurred in these regions. At
passage 10, some clones (8 of 31) still contained the free pCJ32
plasmid, but most of them presented new hybridization profiles (lanes 6 to 9). In clones 10 and 12 (lanes 8 and 9, respectively), the DNA band
corresponding to the 6.5-kbp EcoRI fragment of the free
plasmid (lane 1) was very faintly detected, whereas two additional fragments of 3.6 and 11.9 kbp were clearly seen. In clones 18 and 26 (lanes 6 and 7, respectively), the 4-kbp fragment of the free plasmid
was no longer detected; instead, an additional fragment of
approximately 6.5 kbp was found to be present. Interestingly, in all of
clones 10, 12, 18, and 26, the 9-kbp chromosomal DNA fragment
containing the oriC region was not detected, suggesting that
in these clones, integration of the pCJ32 plasmid may have occurred in
this region. Indeed, hybridization of genomic DNA with an
oriC probe revealed that in clones 10, 12, 18, and 26, the
pCJ32 plasmid had integrated into the spiroplasmal chromosome by
homologous recombination at the oriC region, upstream of the BglII site (ori 1) in clones 18 and 26 and downstream of
this site (ori 2) in clones 10 and 12. The results obtained with clones 10 and 18 are presented in Fig. 1B (lanes 3 and 4) and
2. In clone 18 (Fig. 1B, lane 3), the
9-kbp EcoRI fragment carrying the oriC region in
the wild-type strain (lane 2) was not detected. Instead, the presence
of three fragments of 6.2, 6.45, and 6.5 kbp hybridizing with the
oriC probe indicates that plasmid integration did occur by
recombination at the oriC region, upstream of the
BglII site (Fig. 2A). In clone 10 (Fig. 1B, lane 4), the
detection of three fragments of 3.6, 4, and 11.9 kbp indicates that in
this clone also, plasmid integration occurred within the
oriC region, downstream of the BglII site (Fig.
2B). These results were confirmed by additional Southern blot
hybridizations of NsiI-plus-PstI-restricted DNA (data not shown).
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FIG. 1.
Southern blot hybridization between
EcoRI-restricted DNA extracted from S. citri
transformants at various passages and the pCJ32 (A) and oriC
(B) probes. (A) Lane 1, pCJ32 restricted by EcoRI; lane 2, DNA extracted from untransformed cells; lanes 3 to 5, DNA extracted at
passage 5 from spiroplasmal clones 3, 11, and 30, respectively; lanes 6 to 9, DNA extracted at passage 10 from clones 18, 26, 10, and 12, respectively. (B) Lane 1, pCJ32 restricted by EcoRI; lane 2, DNA extracted from untransformed cells; lanes 3 and 4, DNA extracted at
passage 15 from clones 18 and 10, respectively. Sizes are indicated in
kilobase pairs.
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FIG. 2.
Schematic representation of pCJ32 integration by
recombination at the oriC region. Regions ori 1 and ori 2 represent sequences of the oriC fragment upstream (ori 1)
and downstream (ori 2) of the BglII site of plasmid pBOT1
(33). scm1, 0.9-kbp internal fragment of the
scm1 gene obtained by PCR with primer pair CJ6-CJ17;
tetM, tetracycline resistance gene of Tn916. The
black regions represent pGEM-T Easy vector sequences flanking the
scm1 gene fragment. Bg, BglII; E,
EcoRI; P, PstI; wt. DNA, wild-type DNA. The maps
are not to scale. The size of pCJ32 is 11.55 kbp. (A) Integration into
the ori 1 region; (B) integration into the ori 2 region.
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Five spiroplasmal transformants (clones 11, 23, 24, 28, and 29) that
still carried the pCJ32 plasmid as free extrachromosomal
DNA at passage
10 were further subcultured for five additional
passages and then
tested by PCR for putative integration of the
plasmid by recombination
at the
scm1 gene. According to the results
in Fig.
3, PCR amplification of genomic DNA from
spiroplasmal
transformants with primer pairs CJ26-EV7 and Reverse-CJ5
would
be obtained only if the pCJ32 plasmid had integrated into the
scm1 gene, not if the plasmid was maintained
extrachromosomally
or had integrated into the
oriC region.
In addition, as a result
of plasmid integration, the occurrence of two
incomplete copies
of the
scm1 gene should lead to a
nonmotile phenotype. The results
presented in Fig.
4 show that no amplification was obtained
with
DNA from untransformed GII-3 cells (lanes 2 and 9) or with DNA
from spiroplasmal clones 10 (lanes 4 and 11), 23 (lanes 5 and
12), 24 (lanes 6 and 13), and 28 and 29 (data not shown). In contrast,
the
expected amplification products of 1.4 kbp (1,372 bp) for
primer pair
CJ26-EV7 and 1.4 kbp (1,399 bp) for primer pair Reverse-CJ5
were
obtained in the case of clone 11 (lanes 3 and 10). Therefore,
a broth
culture of this transformant was diluted and plated onto
0.8% agar SP4
plates to determine its motility behavior. Unexpectedly,
most of the
colonies harbored a motile phenotype similar to that
of the wild-type
strain GII-3, while only a few colonies (approximately
5%) showed a
nonmotile phenotype. These observations indicated
that at this stage,
the culture was a mixture of spiroplasma cells
carrying pCJ32 as a free
plasmid (95%) and cells in which pCJ32
had integrated into the
chromosome by recombination at the
scm1 gene (5%). One of
the nonmotile colonies was grown in liquid SP4,
and the resulting
culture was triply cloned (
38) before being
further analyzed
by PCR and Southern blot hybridization. When
plated on agar medium, the
triply cloned culture yielded 100%
colonies having a nonmotile
phenotype. The appearance of these
colonies (Fig.
5B) was identical to that of the
insertional mutant
G540 in which the
scm1 gene was disrupted
by Tn
4001 insertion
(
19) (Fig.
5C). Dark-field
microscopy observation of broth cultures
revealed that this clone, like
the insertional mutant G540, had
retained its helical morphology but,
due to the absence of rotational
movement, was unable of translational
motility in viscous medium.
This spiroplasmal clone was named GII-3 m1.
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FIG. 3.
Schematic representation of pCJ32 integration by
recombination at the scm1 gene. The positions of primers
(notation on figure in parentheses) Reverse (R), CJ5 (5),
CJ6 (6), CJ8 (8), CJ17 (17), CJ26
(26), CJ27 (27), EV7 (ev7), Tet1 (tet1), and Tet2
(tet2) are indicated by short arrows. N, NsiI. For other
abbreviations, see legend to Fig. 2. The maps are not to scale.
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FIG. 4.
PCR amplification of genomic DNA from various
spiroplasmal transformants with primer pairs CJ26-EV7 (lanes 1 to 6)
and Reverse-CJ5 (lanes 8 to 13). Lane 7, 1-kbp DNA ladder; lanes 1 and
8, control without DNA. In lanes 2 to 6 and 8 to 13, target DNAs were
from spiroplasmal clones 10, 11, 23, 24, and untransformed GII-3,
respectively. Sizes are indicated in kilobase pairs.
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FIG. 5.
Colonies of S. citri grown in 0.8% agar SP4
medium for 8 days. (A) Wild-type GII-3; (B) GII-3 m1; (C)
Tn4001 insertional mutant G540.
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Mapping the pCJ32 insertion site in spiroplasmal transformant GII-3
m1.
To further confirm that pCJ32 had integrated into the
spiroplasmal chromosome by homologous recombination at the
scm1 gene, genomic DNA from GII-3 m1 was restricted with
various enzymes and hybridized with the scm1 probe. In Fig.
6, the hybridization patterns of GII-3 m1 DNA are compared to those of
the DNA extracted from untransformed cells. Regardless of the enzymes
used to restrict the DNA (EcoRI, HindIII,
NsiI, or SpeI), two fragments were found to
hybridize with the scm1 probe in the case of GII-3 m1 (Fig. 6, lanes 2, 4, 6, 9, 12, and 15), whereas only one fragment was found
to contain scm1 sequences in the untransformed cells (lanes 1, 3, 5, 8, 11, 14, and 17). Such a duplication of the scm1
sequences is in agreement with the integration of pCJ32 at the
scm1 gene via a single crossover. In addition, sizes of the
restriction fragments hybridizing with the probe fully match those of
the map in Fig. 3, predicted on the basis of recombination between the
scm1 gene fragment carried by the plasmid and the
full-length scm1 gene carried by the chromosome. In
particular, the two EcoRI fragments larger than 12 and 4.1 kbp (Fig. 6, lane 12) were detected neither in the purified plasmid (lane 13) nor in the untransformed cells (lane 11). Also, as expected from the restriction map of Fig. 3,
the 4.1-kbp EcoRI fragment (Fig. 6, lane 12) was also found
in the EcoRI-plus-NsiI double digest of GII-3 m1
DNA (Fig. 6, lane 9). To further characterize the site of plasmid
integration in GII-3 m1, the two regions containing scm1
sequences were amplified by PCR with primer pairs CJ26-EV7 and
Reverse-CJ5, and the amplification products were cloned and sequenced.
Sequence analyses showed that the CJ26-EV7-amplified DNA fragment
contained the scm1 N-terminal sequences (upstream of CJ17)
that are not present in pCJ32 and did not contain the C-terminal end
(downstream of CJ6). In turn, the Reverse-CJ5 fragment was found to
contain the C-terminal end of the scm1 gene but not the N
terminus. The occurrence of these two truncated copies of the
scm1 gene in the chromosomal DNA of GII-3 m1 indicates that
in this transformant, the pCJ32 plasmid has integrated into the
spiroplasmal chromosome by homologous recombination at the
scm1 gene, via a single crossover. To assess the stability
of plasmid integration in vitro, the scm1-disrupted mutant
was propagated in liquid medium with or without tetracycline for more
than 100 generations and then plated on 0.8% agar plates. No reversion
to the motile phenotype was noted, regardless of the presence or
absence of tetracycline as the selection pressure. Of the more than
1,000 colonies observed, none had spiroplasmas with a motile phenotype.
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FIG. 6.
Southern blot hybridization of genomic DNA from S. citri GII-3 and GII-3 m1 with the scm1 probe. DNAs from
GII-3 (lanes 1, 3, 5, 8, 11, 14, and 17) and GII-3 m1 (lanes 2, 4, 6, 9, 12, 15, and 18) were restricted by HindIII (lanes 1 and 2), HindIII plus NsiI (lanes 3 and 4),
NsiI (lanes 5 and 6), NsiI plus EcoRI
(lanes 8 and 9), EcoRI (lanes 11 and 12), SpeI
(lanes 14 and 15), and EcoRV (lanes 17 and 18). Plasmid
pCJ32 (lanes 7, 10, and 13) was restricted by NsiI (lane 7),
NsiI plus EcoRI (lane 10), and EcoRI
(lane 13). Sizes are indicated in kilobase pairs.
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Experimental transmission of GII-3 m1 to periwinkle plants.
The motility mutant GII-3 m1 was tested for its ability to be
transmitted by the leafhopper vector and to induce symptoms in the host
plant. A culture of GII-3 m1 was microinjected into 50 leafhoppers as
described in Materials and Methods, and the injected insects were caged
on five separate periwinkle plants (10 insects per plant). After a
2-week period, the insects were removed and tested for transmission
through a Parafilm membrane. Plant symptoms were monitored for 6 weeks
after insect removal. Multiplication of S. citri in the
injected leafhoppers and in the plants was followed by determining the
number of CFU per insect or per milligram of midribs. For each
experiment, wild-type S. citri GII-3 and S. citri-free SP4 medium were used as positive and negative controls,
respectively. Spiroplasmal transformant clone 18, in which the pCJ32
plasmid has integrated into the oriC region, was also
tested. The results are summarized in Table
1. As expected, in the case of the
wild-type strain GII-3 as well as with clone 18, severe symptoms were
produced within 2 weeks after insect removal; in agreement with these
results, transmission assays through Parafilm membranes were all
positive. Interestingly, similar results were obtained with the
scm1-disrupted mutant GII-3 m1. Four of five periwinkle
plants (P1, P2, P4, and P5) showed symptoms characteristic of the
disease; only one (P3) was symptomless. This absence of symptoms could
be explained by the fact that plant P3 was uninfected; most (7 of
10) of the insects fed on this plant died 1 day after
injection. Also, Table 1 shows that the three surviving leafhoppers
failed to transmit spiroplasmas through the Parafilm membrane,
suggesting that these three insects were probably poorly infected.
Despite this particular case, the results clearly show that the
motility mutant GII-3 m1 was transmitted by the leafhopper vector to
the other four periwinkle plants, in which it induced symptoms within 2 weeks after transmission. Determinations of CFU showed that in the
injected leafhoppers, the motility mutant GII-3 m1 multiplied to
approximately the same titer (1.4 × 106 CFU/insect)
as did the wild-type strain GII-3 (1.5 × 106
CFU/insect) and clone 18 (1.7 × 106 CFU/insect). In
the plants, spiroplasma titers in the symptomatic leaves were also
found to have similar values: 5.7 × 103, 9.1 × 103, and 6.8 × 103 CFU/mg of midribs for
GII-3 m1, GII-3, and clone 18, respectively. Symptoms in these plants
(asymmetric and chlorotic young leaves, chlorosis on older leaves,
stunting, and eventually lethal yellowing) were identical to those
shown by plants infected with clone 18 or with the wild-type strain
GII-3. To further confirm that symptom expression was due to the
multiplication of mutant GII-3 m1, spiroplasmas were cultured from
GII-3 m1-infected plants and dilutions of the culture were plated on
0.8% agar medium. All colonies expressed a nonmotile phenotype,
indicating that no contaminant or no revertant spiroplasma was present.
In addition, Southern blot hybridization of genomic DNA with the
scm1 probe proved the gene organization around the
scm1 gene to be identical to that of the injected
spiroplasma (data not shown).
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Experimental transmission of S. citri GII-3,
GII-3 m1, and GII-3 ori (clone 18) to periwinkle (Catharanthus
roseus) by the leafhopper vector C. haematoceps
|
|
| |
DISCUSSION |
In this study, a motility mutant of S. citri was
generated by disruption of the scm1 gene through homologous
recombination. Production of mutants by gene disruption or allelic
exchange, both of which are dependent on homologous recombination, is
crucial to assess the role of individual genes in various processes
such as pathogenesis. It has been extensively used in many bacteria, including the mollicutes A. laidlawii (11) and
M. genitalium (10). However, the homologous
recombination process usually requires the RecA protein (22,
28), which is probably absent from S. citri (26,
27). Therefore, in the absence of a functional RecA protein, the
opportunity for homologous recombination was expected to be severely
limited when nonreplicating DNA was used. Indeed, when spiroplasma
cells were transformed with the suicide plasmid pKTM1, no
tetracycline-resistant transformants were obtained, indicating that no
recombination had occurred between the plasmid carrying the
scm1 gene fragment and the spiroplasmal chromosome. Similarly, it was shown that the recA mutant strain 8195 of
A. laidlawii could not be transformed with the
nonreplicative plasmid pKA8195, whereas the wild-type strain JA1 could
be (11). In S. citri, plasmid integration by
homologous recombination at the scm1 gene could be obtained
only when spiroplasma cells were transformed with the replicative
plasmid pCJ32. This plasmid construct was based on the oriC
plasmid pBOT1, which was shown to replicate in S. citri as a
free plasmid before it integrates into the spiroplasmal chromosome by
recombination at the oriC region (33). Plasmid integration was thought to be due to the incompatibility of
oriC plasmids. It is known that in gram-positive bacteria,
the presence of plasmids containing dnaA boxes strongly
inhibits bacterial growth. As a result, one copy of oriC
plasmid can barely coexist with the chromosome; the plasmid either is
lost or integrates into the bacterial chromosome (29).
Because general recombination is most efficient when it operates on
long stretches of highly homologous DNA, inserting the scm1
gene fragment within the oriC region was expected to
decrease the frequency of recombination in this region. In spite of
this cloning strategy, the results showed that most of the
recombination events leading to pCJ32 integration still occurred at the
oriC region, not only within the 1.3-kbp ori 1 but also
within the 0.6-kbp ori 2 fragment (Fig. 2), the size of which was still
much greater than the minimal extent of homology (estimated to 70 bp)
necessary to promote homologous recombination in Bacillus
subtilis (20). Interestingly, in transformant GII-3 m1,
plasmid integration did not occur at the oriC region but
instead occurred within the scm1 gene, leading to the
structure expected for the accurate product of homologous recombination between the scm1 gene fragment carried by pCJ32 and the
full-length scm1 gene of the spiroplasmal chromosome.
Indeed, disruption of the scm1 gene led to a nonmotile
phenotype, indicating that neither of the two truncated copies of the
scm1 gene, resulting from plasmid integration, was
translated to a functional polypeptide. The phenotype of the
scm1-disrupted mutant was identical to that of the
scm1 mutant G540 obtained by Tn4001 mutagenesis
(19). Due to the absence of rotational movement, these two
mutants are incapable of translational motility in viscous medium and
form compact, sharp-edged colonies, even when plated in low-agar medium.
This study and others from our laboratory (17) demonstrate
gene disruption through homologous recombination in S. citri. These results highlight the great potential of replicative
oriC plasmids to promote homologous recombination in
S. citri, an organism which probably lacks the RecA protein.
The use of replicative oriC plasmids increases the time over
which recombination can occur (compared to nonreplicative plasmids); in
addition, the incompatibility of oriC plasmids is used as a
selection pressure for plasmid integration. However, our results showed
that in most of the transformants, plasmid integration occurred at the
oriC region rather than at the scm1 gene. In
other words, recombination at oriC was much more frequent
than recombination at the scm1 gene. In the plasmid vector
pBOT1, the oriC fragment comprises the dnaA gene
flanked by two dnaA box regions. However, we have shown that
the dnaA gene and the dnaA box region upstream of
it are not required for plasmid replication, suggesting that in this plasmid, the replication origin could be reduced to the dnaA
box region located downstream of the dnaA gene
(33). Assuming that recombination frequency is, in part, a
function of the length of homologous sequences, the use of
oriC plasmids with a replication origin reduced to the
shortest fragment still containing the dnaA boxes should
decrease the frequency of plasmid integration at the oriC
region and, in turn, increase the frequency of recombination within the
gene of interest. In addition, it would be useful to study
systematically the effect of length of homology on recombination in
S. citri and determine whether the frequency of
recombination is gene dependent. Whether allelic exchange via a double
crossover could be obtained by using extremely long regions of homology also must be determined. For most bacteria, an understanding of gene
function is dependent on the availability of relevant mutants. The
ability to construct site-specific mutations in S. citri
will provide novel insights into the biology of this plant-pathogenic mollicute.
In many pathogenic bacteria, motility and chemotaxis are major factors
of virulence, allowing the bacterial cells to reach the site of
infection and invade the host tissues (30, 39). In this
respect, it was hypothesized that the motility of S. citri might play a role in the spiroplasma-host interactions. In particular, it was proposed that motility, by facilitating dispersal in the host,
would be one of the pathogenicity factors developed by spiroplasmas (21). To test this hypothesis, the scm1-disrupted
motility mutant was experimentally transmitted to periwinkle plants
through leafhopper vectors injected with the mutant. We found that the
motility mutant multiplied in the insects and was efficiently
transmitted to periwinkles, in which it induced symptoms similar to
those obtained with control spiroplasma strains: the wild-type strain
GII-3 and clone 18 in which pCJ32 has integrated into the
oriC region. These results suggest that S. citri
motility, and particularly the rotational movement responsible for
translational motility in viscous medium that is lost in the
scm1 mutant, is not required for the spiroplasma to reach
and invade the salivary glands. In addition, the occurrence of severe
symptoms in the GII-3 m1-infected periwinkles suggests that the
scm1 gene product, and hence motility, is not essential for
pathogenicity. This is in good agreement with the fact that the
nonhelical, nonmotile S. citri ASP1 was isolated from a
symptomatic sweet orange tree and found to cause symptoms when
experimentally transmitted to broad bean seedlings (37). It
is noteworthy that no motile revertants were isolated, either from
GII-3 m1-injected insects or from GII-3 m1-infected periwinkles,
suggesting that motility might not be an advantage for S. citri propagation, not only in vitro but also in the insect vector
and in the host plant. Furthermore, both in insects and in plants, the
titer of GII-3 m1 was found to be not significantly lower than the
titers of the motile, control strains. However, the possibility that
the motility behavior of the scm1-disrupted mutant in its
hosts might be different from that observed in vitro cannot be
excluded. According to the life cycle of S. citri,
spiroplasmas ingested from plant phloem face two barriers in the insect
vector, the gut epithelium and the salivary gland membrane (13,
23). Comparison of transmissible and nontransmissible lines of
S. citri BR3 led to the characterization of a gene encoding
an adhesin-like protein. However, the involvement of this protein in
spiroplasma-insect vector interactions remains to be demonstrated
(46). In our studies, transmission assays in which
spiroplasmas were injected directly into the insect hemolymph showed
that the scm1-disrupted mutant was not affected in its ability to cross the salivary gland membrane to reach the saliva duct.
Whether this mutant is able to cross the gut epithelium barrier when
the leafhopper vector is fed on scm1 mutant-infected plants
is currently under investigation.
| |
ACKNOWLEDGMENTS |
We are grateful to our colleagues J. M. Bové for
critical reading of the manuscript and P. Gaurivaud for helpful
discussions. We also thank P. Bonnet and J. B. Reynaud for growing
plants and insects.
This work was financially supported in part by an AIP Microbiologie
grant from INRA.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Biologie Cellulaire et Moléculaire, INRA et Université
Victor Segalen Bordeaux 2, B.P. 81, 33883 Villenave d'Ornon Cedex,
France. Phone: (33) 5.56.84.31.51. Fax: (33) 5.56.84.31.59. E-mail:
renaudin{at}bordeaux.inra.fr.
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Abstract
Introduction
