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Applied and Environmental Microbiology, October 1998, p. 3948-3953, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Closely Related Plasmid Replicons Coexisting in the Phytopathogen
Pseudomonas syringae Show a Mosaic Organization of the
Replication Region and Altered Incompatibility
Behavior
Ane
Sesma,1
George W.
Sundin,2 and
Jesús
Murillo1,*
Laboratorio de Patología Vegetal,
Escuela Técnica Superior de Ingenieros Agrónomos,
Universidad Pública de Navarra, 31006 Pamplona,
Spain,1 and
Department of Plant
Pathology and Microbiology, Texas A&M University, College Station,
Texas 77843-21322
Received 2 March 1998/Accepted 3 July 1998
 |
ABSTRACT |
Many Pseudomonas syringae strains contain native
plasmids that are important for host-pathogen interactions, and most of
them contain several coexisting plasmids (pPT23A-like plasmids) that cross-hybridize to replication sequences from pPT23A, which also carries a gene cluster coding for the phytotoxin coronatine in P. syringae pv. tomato PT23. In this study, three
functional pPT23A-like replicons were cloned from P. syringae pv. glycinea race 6, suggesting that the compatibility
of highly related replicons is a common feature of P. syringae strains. Hybridization experiments using three
separate incompatibility determinants previously identified from pPT23A
and the rulAB (UV radiation tolerance) genes showed that
the organization of the replication region among pPT23A-like plasmids from several P. syringae pathovars is poorly
conserved. The putative repA gene from four pPT23A-like
replicons from P. syringae pv. glycinea race 6 was
amplified by using specific primers. The restriction profiles of the
resulting PCR products for the race 6 plasmids were more similar to
each other than they were to that of pPT23A. These data, together with
the existence of other cross-hybridizing DNA regions around the
replicon among the race 6 pPT23A-like plasmids, suggest that some of
these plasmids may have originated from duplication events. Our results
also imply that modifications of the repA sequences and the
poor conservation of putative maintenance determinants contribute to
the suppression of incompatibility among members of the pPT23A-like
family, thus enhancing the genomic plasticity of P. syringae.
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INTRODUCTION |
Strains of the phytopathogenic
bacterium Pseudomonas syringae cause economically important
losses to crop productivity by inciting disease and, in some cases, by
catalyzing frost injury. Isolates of this species are grouped in 46 pathovars depending on the host range. Most P. syringae
strains contain one or more indigenous plasmids of variable size (1 to
>100 kb), some of which are known to be conjugative (10,
29). In many cases, these plasmids are important or essential for
host-pathogen interactions, since they contain genes involved in
pathogenicity (21), biosynthesis of extracellular virulence
factors such as ethylene, indoleacetic acid, and the phytotoxin
coronatine (3, 8, 16, 24), determination of host range by
avirulence genes (33), competitive fitness (30),
resistance to antibacterial compounds, such as copper, streptomycin,
and trimethoprim (9), and/or resistance to UV radiation
(32). An interesting facet of P. syringae
plasmid biology is the existence of repeated sequences or areas of
homology among different native plasmids of a given strain, which, in
the best characterized case of plasmids pPT23A (100 kb) and pPT23B (83 kb) from P. syringae pv. tomato PT23, account for an
estimated 74% of their total DNA (23).
A 9.2-kb KpnI fragment from pPT23A that could autonomously
replicate in P. syringae has been previously cloned and
characterized, and the minimal region that retained the capacity to
replicate (oriV-pPT23A) has been defined to a 1.6-kb
fragment (23). Hybridization experiments have shown that
oriV-pPT23A is highly conserved in pPT23B and that as many
as six plasmids in a given P. syringae strain
cross-hybridize with these sequences. Due to the possibility that they
originate from a common ancestor, the name pPT23A-like was proposed to
designate collectively the family of plasmids that show
cross-hybridization with oriV-pPT23A (13). The
nucleotide sequence of oriV-pPT23A (13) has been
shown to contain a gene, repA, that codes for a protein
essential for replication which is highly similar to the replication
proteins of pTiK12, from Thiobacillus intermedius, and
ColE2-like plasmids, among others. The identification of other
maintenance determinants of pPT23A was based on the fact that most of
them are able to displace their parent plasmids when both are present
in the same cell and selection is applied for the cloned
determinant. In this way, it was shown that pAKC contains three
determinants (IncA, IncB, and IncC) that exert a strong incompatibility
with pPT23A: IncA is represented by a 400-bp
PstI-EcoRI fragment upstream of
oriV-pPT23A, IncB overlaps oriV-pPT23A, and
IncC is included in a 0.8-kb EcoRI-KpnI fragment located at one end of pAKC. The defined IncC is
part of a putative partition system that increases the stability of the
cloned oriV-pPT23A (6), while the functional
significance of the IncA determinant is currently unknown. Partial
sequencing has also shown that an operon involved in resistance to UV
radiation (rulAB genes), which could be conferring a
selective advantage for the maintenance of pPT23A, is located
immediately downstream of oriV-pPT23A and preceding IncC.
It is generally accepted that plasmids that contain related sequences
functioning in replication or partition cannot be stably maintained in
growing populations of bacteria due to incompatibility effects
(1, 25, 26). By contrast, the homology of
oriV-pPT23A sequences among coexisting plasmids within
P. syringae represents a phenomenon that has not been
described for other organisms. The RepA protein of the related ColE2
plasmids functions in trans as an activator of replication
by binding to the origin sequence in a plasmid-specific manner and
synthesizing a primer RNA for initiation of DNA synthesis by DNA
polymerase I at the origin (14). Thus, opportunities for the
evolution of compatibility among pPT23A-like plasmids having highly
homologous RepA proteins may involve sequence divergence within
respective repA and ori sequences maintaining a
plasmid-specific method of binding. Alternatively, the observed
compatibility could be dependent on the absence or modification of
other Inc determinants, such as IncA and IncC. We are interested in
identifying the genetic and biochemical mechanisms enabling such
related plasmids to coexist and also in determining the effect of
homology of oriV-pPT23A sequences on the horizontal exchange
of plasmids among P. syringae strains. In this study, we examined the compatibility of native pPT23A-like plasmids from P. syringae pathovars with pPT23A and determined the
distribution of specific Inc sequences among these plasmids. To
further analyze the compatibility phenomenon of coexisting pPT23A-like
plasmids, we cloned and studied the functionality and
sequence divergence of the oriV sequences from three
pPT23A-like plasmids from P. syringae pv. glycinea race
6.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth conditions.
Escherichia coli DH5
(Life Technologies, Inc.,
Gaithersburg, Md.) was used for cloning procedures. P. syringae pathovars tomato (strains PT17, PT23, PT30
[2], and B120), apii (strain 1089-5), mori (strain
0782-30), morsprunorum (strain 0782-28), and savastanoi (strain 0485-9)
were obtained from D. Cooksey (University of California at Riverside).
P. syringae pathovars tomato (strain DC3000)
(12) and glycinea (races 4, 5, and 6) (18) were
obtained from N. T. Keen (University of California at Riverside);
and P. syringae pathovars pisi (strain PN8)
(22) and phaseolicola (strain 1302A) were from J. D. Taylor (HRI, Wellesbourne, United Kingdom). The plasmid content of some
of these strains has been reported earlier (23). Native
plasmids were designated by using an acronym derived from the strain
name and a letter in alphabetical order starting from the largest
plasmid. P. syringae pv. syringae FF5 is a plasmidless
strain that shows a high efficiency of electroporation (31).
Plasmids pBluescript SK+ (Stratagene, La Jolla, Calif.) and
pMTL24 (7), which contains a symmetric polylinker, were used for cloning purposes. Plasmid pK184 (Kmr) (15),
which does not replicate in Pseudomonas, was utilized for
testing the functionality of putative origins of replication. pAKC
contains a 9.2-kb KpnI fragment from the largest native
plasmid of strain PT23 (pPT23A) cloned in pK184 and can replicate
autonomously in P. syringae (23).
E. coli was grown in Luria broth (LB) medium at 37°C, and
P. syringae was cultivated in King's medium B (KMB)
(
19) or in
LB medium at 28°C. When necessary, media were
supplemented with
the following antibiotics at the indicated
concentrations: ampicillin,
100 µg/ml; carbenicillin, 100 µg/ml;
and kanamycin, 25 µg/ml.
Genetic and molecular biology techniques.
Standard molecular
biology techniques were used (27). Plasmid DNA
minipreparations were prepared from 1.5 ml of an overnight culture in
KMB by using a modified alkaline lysis procedure (34). In
some instances, plasmids were purified by isopycnic centrifugation in
CsCl (27). Intact plasmid DNA was separated by
electrophoresis on 0.6% agarose (Pronadisa; Hispanlab S.A., Madrid,
Spain) gels in 1× Tris acetate-EDTA (TAE) buffer at 3.2 V/cm for 5 to
6 h at room temperature or 16 to 17 h at 4°C. Plasmids were
introduced into Pseudomonas by electroporation
(17). Cloning of DNA fragments was done essentially as
described previously (11).
Amplification of DNA by the PCR was performed with primers RE1.1
(5'-AGTGACGACAAAACCGC-3') and M553
(5'-GAGAATTCCGTGAGGATGTG-3'),
which flank a 933-bp fragment
of the
repA gene from pPT23A corresponding
to nucleotides
159 to 1093 of the coding region (
13). Native
plasmids used
as templates for PCR were individually excised from
agarose gels and
purified by using 0.2-µm Nanosep MF columns as
described in the
manufacturer's instructions (Pall Filtron, Northborough,
Mass.).
Conditions for amplification were the following: 1.7 mM
MgCl
2, 125 µM deoxynucleoside triphosphates, 0.4 µM
each primer,
and 0.05 U of
Taq polymerase/µl (BioTaq;
Bioline United Kingdom
Ltd., London, United Kingdom). The amplification
was done on a
Linus Autocycler Plus (Corbett Research, Sydney,
Australia) with
a cycle of 94°C for 2 min, followed by 30 cycles of 1 min per
step at 94, 50, and 72°C, and a final extension step of 10 min
at 72°C. PCR products digested with
HaeIII were
separated on 3%
MS8 agarose (Hispanlab S.A.) gels in 1× TAE buffer.
Fragments to be used as probes were cloned separately in pBluescript
SK
+ or pK184, excised from the vector, and separated in
low-melting-point
agarose before being labeled with
[
-
32P]dCTP (
27). DNA separated by
electrophoresis and transferred
to nylon membranes (Hybond-N+;
Amersham) was hybridized at 42°C
in 5× SSC (1× SSC is 0.18 M NaCl,
10 mM NaH
2PO
4, and 1 mM EDTA
[pH 7.7])-50%
formamide-20 mM NaPO
4 (pH 7.0)-1× Denhardt's
solution-0.1
mg of herring-sperm DNA ml
1
(
20). After hybridization, the blots were washed twice, 15
min each, at 42°C in 2× SSC-0.1% sodium dodecyl sulfate and
exposed
while damp to X-ray film (Biomax; Kodak, Rochester, N.Y.) for
3 to 48 h at
80°C with intensifying screens. When necessary,
DNA
fragments were labeled with digoxigenin and used as hybridization
probes as described in the manufacturer's instructions (Boehringer
GmbH, Mannheim, Germany).
Incompatibility assays.
The incompatibility between pAKC and
related plasmids was assayed by a qualitative test essentially as
described previously (25). Briefly, plasmid pAKC was
introduced by electroporation, and 3 to 14 transformants for each
strain resulting from at least three independent electroporations were
selected in KMB plus kanamycin. To evaluate a possible partial
incompatibility of a native plasmid with pAKC, isolated colonies of
each transformant were serially transferred in KMB plus kanamycin up to
four times. The plasmid profile of the clones purified from the
selection plate and after the fourth transfer in selective media was
examined.
Cloning of origins of replication from native plasmids.
Total plasmid DNA from P. syringae pv. glycinea race 6 purified by CsCl gradient centrifugation was digested to completion with KpnI and ligated en masse to the E. coli
vector pK184, which cannot replicate in Pseudomonas
(15). KpnI cuts P. syringae DNA
at a low frequency, and previous experiments showed that the homology
with oriV-pPT23A was located in KpnI fragments
larger than 7 kb (data not shown). The ligation mixture was purified by
phenol-chloroform extraction (27) and resuspended in sterile distilled water, and aliquots were transformed in P. syringae pv. apii 1089-5, which shows a high frequency of
electroporation. Plasmid DNA was extracted from transformants growing
in KMB plus kanamycin and transformed into DH5, to analyze the
cloned inserts by restriction digestion, and into the plasmidless
strain P. syringae pv. syringae FF5, to confirm
the autoreplicative ability of the recombinant plasmids. We
confirmed that the cloned inserts had arisen from race 6 plasmid DNA by
using the cloned inserts, or internal fragments thereof, as
hybridization probes against double digestions of the corresponding
DNAs and comparing the hybridization patterns (see Fig. 3; also data
not shown).
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RESULTS |
Incompatibility of pAKC with native plasmids from several
P. syringae strains.
The presence of closely
related plasmids among P. syringae strains could limit
the potential for horizontal spread. To obtain a measure of the
relatedness of plasmids among a range of P. syringae strains, pAKC was introduced by
electroporation and the resulting transformants were examined to
observe the extent of plasmid curing resulting from incompatibility.
The strains tested contained one to six plasmids with homology to
oriV-pPT23A (Fig.
1 and
2). The Km
r
transformants contained pAKC and, in most cases, one or two novel
bands
of different intensities that showed homology to pAKC (Fig.
1; also data not shown): this was
probably due to the formation
of multimers of the incoming plasmid. All
14 transformants obtained
from
P. syringae pv. glycinea
race 4 lost plasmids pR4C, pR4D,
and pR4F, two of which (pR4C and pR4F)
cross-hybridized to
oriV-pPT23A.
As expected, since pAKC
contains
oriV-pPT23A, the pPT23A plasmid
was evicted from
all of the PT23 transformants (Fig.
1). In 5
of 14 transformants
analyzed, pPT23B was also evicted, which could
be due to the fact that
pPT23A and pPT23B have highly homologous
origins of replication
(
23). A plasmid with a size similar to
that of pPT23A that
cross-hybridizes with
oriV-pPT23A (Fig.
2)
was also
evicted in all cases from
P. syringae pv. tomato
PT17
and PT30. Strains PT17, PT23, and PT30 were all isolated in
California,
display very similar plasmid profiles, and are probably
variants
of the same strain that differ only in their plasmid
content (
2).
Immediate eviction of the
incompatible plasmids was observed,
and in all of the cases described
above, plasmids were already
absent from colonies cultured directly
from the selection plates.
With the remaining strains (
P. syringae pathovars apii 1089-5,
glycinea race 6, mori
0782-30, morsprunorum 0782-28, and tomato
B120 and DC3000),
we could not observe specific curing of any
of the native plasmids,
even after four successive transfers in
KMB plus kanamycin.
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FIG. 1.
Incompatibility between pAKC and related native plasmids
from P. syringae strains. Panels show plasmid profiles
of each strain before (left lanes) and after (right lanes) introduction
of pAKC (arrowhead). Asterisks indicate plasmids hybridizing to the
minimal origin of replication from pPT23A (see Fig. 2), and arrows
indicate evicted plasmids. Abbreviations for P. syringae pathovars: Pap, apii; Pgy, glycinea; Pmo, mori; Pmp,
morsprunorum; Pto, tomato. Short horizontal lines indicate the
positions of chromosomal and/or linear plasmid DNA. Gels were scanned
(Umax Vista-S6) and labeled by using PowerPoint and a PC Compaq 575e.
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Conservation of incompatibility determinants and the
rulAB genes among P. syringae native
plasmids.
pAKC contains three regions that demonstrate strong
incompatibility with pPT23A, one of which (IncB) includes the minimal fragment able to replicate autonomously (13). The observed
lack of incompatibility between pAKC and other plasmids of the
pPT23A-like family suggest that plasmids related to pPT23A could have
undergone evolutionary changes in the three Inc regions that allow for
their stable coexistence. We therefore analyzed by hybridization the conservation of the determinants linked to oriV-pPT23A among
plasmids of the 10 strains included in the earlier incompatibility
assay (Fig. 1) plus 4 other P. syringae strains. We
used DNA fragments from the three defined Inc regions and from the
rulAB operon, which is located adjacent to
oriV-pPT23A (13) (see Fig. 2), as hybridization
probes.
All of the strains analyzed were shown to contain one to six plasmids
with homology to the IncB probe (Fig.
2).
Based on the
intensity of the hybridization signals, IncA-hybridizing
sequences
were detected in three plasmids in
P. syringae pv. glycinea race
6 and in two plasmids in
P. syringae pv. tomato PT17, PT23, and
PT30, while
rulAB-hybridizing sequences were detected in three
plasmids
in
P. syringae pv. glycinea race 4. In contrast to the
situation regarding IncB, the IncA and
rulAB probes showed
strong
hybridization with only one plasmid in five and nine of the
strains,
respectively, although a few of them also contained one to two
plasmids with weak hybridization to the probes. Only five of the
strains contained a plasmid that hybridized to the IncC probe,
despite
the fact that they contained another plasmid(s) of the
pPT23A-like
family. In all cases, hybridization to the IncA, IncC,
or
rulAB probe was associated only with plasmids that also
hybridized
to the IncB probe. These results suggest that except for the
conservation
of IncB, the replication region among plasmids of the
pPT23A-like
family varies. This is further supported by the fact that
only
one plasmid in strains
P. syringae pv. glycinea
race 4 and
P. syringae pv. tomato PT17, PT23, and PT30
cross-hybridized to all
four probes.
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FIG. 2.
Conservation of incompatibility determinants and
rulAB genes in different P. syringae
pathovars. (a) Structure of the 9.2-kb KpnI insert of pAKC.
Arrows indicate the approximate locations of the rulAB
genes, as defined by partial sequencing, and the gene for the putative
replication protein (repA). Thick lines under the map show
the extent of the incompatibility regions. Solid rectangles labeled A,
B, R, and C indicate the restriction fragments used as specific probes
for IncA, IncB (minimal origin of replication), rulAB, and
IncC, respectively. B, BamHI; E, EcoRI; K,
KpnI; P, PstI. (b) Diagram showing the plasmid
profiles of different P. syringae strains. Letters
above plasmid bands indicate strong or weak (in brackets)
cross-hybridization with the probes labeled as shown in panel a.
Asterisks indicate plasmids evicted by pAKC in the incompatibility
assay (see Fig. 1). Abbreviations for P. syringae
pathovars: Pap, apii; Pgy, glycinea; Pmo, mori; Pmp, morsprunorum; Pph,
phaseolicola; Ppi, pisi; Psv, savastanoi; Pto, tomato. Numbers on the
right indicate in kilobases the sizes of the plasmids from strain
PT23.
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Cloning of origins of replication from P. syringae
pv. glycinea race 6.
P. syringae pv. apii 1089-5, used
as a host for cloned origins of replication, contains a single
plasmid of 80 kb (p1089A). Eleven Kmr transformants
obtained contained p1089A and an additional plasmid of 11 to 18 kb (data not shown). Total plasmid DNA isolated from these colonies was
individually transformed to E. coli DH5 and analyzed
by restriction digestion. A total of four different plasmids, pORI601 to -604, were identified on the basis of their restriction profiles with KpnI-EcoRI and
KpnI-HindIII (Fig.
3A; also data not shown). These
plasmids were individually purified from E. coli and found
to replicate autonomously in the plasmidless strain P. syringae pv. syringae FF5, which indicates that they contain a functional origin of replication. Plasmids pORI601, pORI602, and pORI603, but not pORI604, cross-hybridized with the 0.8-kb EcoRI fragment from pAKC (Fig. 3B). This fragment contains
the first 528 nucleotides of the coding sequence of the putative RepA protein of oriV-pPT23A and 308 upstream nucleotides that are
also required for replication (13, 23).
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FIG. 3.
Hybridization of the cloned origins of replication from
P. syringae pv. glycinea race 6 with
oriV-pPT23A. (A) DNAs from pORI601 (lane 1), pORI602 (lane
2), pORI604 (lane 3), and pORI603 (lane 4), and total plasmid DNA from
P. syringae pv. glycinea race 6 (lane 5) were digested
with KpnI and EcoRI and separated on an agarose
gel. (B) A gel similar to the one shown in panel A was subjected to
Southern blot analysis by using a 0.8-kb EcoRI fragment from
pAKC as a specific oriV probe from pPT23A. Lane  , lambda
DNA digested with HindIII. The gel and autoradiogram
were scanned (Umax Vista-S6) and labeled by using PowerPoint and a PC
Compaq 575e.
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To identify the replication determinants in pORI601, -602, and -603, the plasmids were partially digested with
Sau3AI and
separated in a low-melting-point agarose gel and fragments 2 to
3 kb in size were cloned in pMTL24, producing plasmids pORI605,
-606, and
-607, respectively. These three plasmids contained approximately
3-kb inserts, replicated autonomously in
P. syringae
pv. syringae
FF5, and cross-hybridized with the 0.8-kb
EcoRI
fragment from
pAKC (data not shown). These results indicate that the
DNA homologous
to
oriV-pPT23A in race 6 represents, at least
in some cases, functional
origins of replication.
Plasmids pORI601, -602, and -603 shared many cross-hybridizing
restriction fragments besides the origin of replication (data
not
shown). In consequence, and to ascertain that the cloned origins
of
replication were derived from different native plasmids, we
performed
Southern hybridization analysis with selected non-cross-hybridizing
restriction fragments from each plasmid as probes (Fig.
4). Probes
derived from pORI602 and
pORI603 hybridized strongly to plasmids
pR6E and pR6D, respectively,
confirming that they originated from
different native plasmids. In
longer exposures, the probe from
pORI602 also hybridized to plasmids B,
C, and D, and the probe
from pORI603 hybridized to plasmids B, E, and
F. The probe derived
from plasmid pORI601, however, showed strong
hybridization to
both pR6C and pR6F and weak hybridization to pR6B and
pR6E. These
results indicate that DNA adjacent to the replication
region is
conserved among most of the pPT23A-like
plasmids from
P. syringae pv. glycinea race 6.
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FIG. 4.
Assignment of cloned pPT23A-like origins of replication
to native plasmids in P. syringae pv. glycinea race 6. Native plasmids from P. syringae pv. glycinea race 6 were separated on an agarose gel (lane 1) and hybridized by using as
probes a 0.8-kb EcoRI fragment from pORI601 (lane 2), a
0.8-kb HindIII fragment from pORI602 (lane 3), or a
0.6-kb EcoRI-HindIII fragment from pORI603
(lane 4). Autoradiograms are overexposed to show weaker hybridization.
Plasmid pR6G did not cross-hybridize to any of the probes and was not
included to reduce the size of the figure. The gel and autoradiograms
were scanned (Umax Vista-S6) and labeled by using PowerPoint and a PC
Compaq 575e.
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Plasmids pORI602 and pORI604 were introduced by electroporation
in
P. syringae pv. glycinea race 6. The results of
four independent
transformations with pORI602 led to the curing
of pR6G (8 kb)
in three cases and in one case of pR6E (70 kb), its
parental plasmid.
On the other hand, pORI604 caused the eviction of
pR6G from the
five transformants analyzed. Since pR6G is the only
plasmid from
P. syringae pv. glycinea race 6 that does
not show homology to
oriV-pPT23A, it is possible that
pORI604 contains the origin of
replication from this plasmid. Despite
numerous attempts, we were
unable to obtain transformants containing
pORI601 or pORI603.
PCR amplification of pPT23A-like replication regions.
To study
the relatedness of the replication regions of pPT23A-like plasmids from
P. syringae pv. glycinea race 6, we examined the
restriction profile of PCR products specifically derived from them.
Using primers RE1.1 and M553, we obtained an amplified product of ca.
900 bp in all cases. Reproducible HaeIII restriction
patterns were generated from the 900-bp PCR product obtained from
plasmids pAKC, pORI601 and pORI603, and with the native plasmids pR6B, pR6C, and pR6F (Fig. 5). All of the
plasmids displayed differential restriction patterns, although the
patterns of the plasmids from race 6 were more similar to each other
than they were to the pattern of pAKC. Taking into account the fragment
order in oriV-pPT23A, the differences in the restriction
pattern of the amplified products could be explained by changes in at
least five positions distributed along the entire sequence of this
region. The comparison of the restriction patterns also suggests that
pORI601 originated from pR6C. No specific amplification products
were obtained with pR6A and pORI602, and no reproducible results
were observed with pR6D and pR6E.
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FIG. 5.
Diversity of restriction patterns among replication
origins related to oriV-pPT23A in P. syringae pv. glycinea race 6. PCR products digested with
HaeIII were separated on a 3% agarose gel. The primers
flank a 933-bp fragment of the coding region of the putative
replication protein of oriV-pPT23A. Lanes: 1, pR6B; 2, pR6C;
3, pR6F; 4, pORI601; 5, pORI603; 6, pAKC. Numbers on the right indicate
the sizes in base pairs of the restriction fragments of pAKC. The gel
was scanned (Umax Vista-S6) and labeled by using PowerPoint and a PC
Compaq 575e.
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DISCUSSION |
Many P. syringae strains contain two or more
plasmids with homology to oriV-pPT23A (Fig. 2)
(23), suggesting that they could have arisen by duplication
of preexisting plasmids. The presence of cross-hybridizing origins of
replication in up to six plasmids of the same cell could be explained
if (i) the origins were no longer functional due to deletion or
mutation, (ii) these plasmids contained another functional origin of
replication, or (iii) the plasmids had evolved mechanisms that allow
them to escape incompatibility. Three cloned origins derived from
different plasmids from P. syringae pv. glycinea race 6 showed similarity to oriV-pPT23A (Fig. 3B), which suggests
that, at least in some cases, this similarity identifies functional
origins of replication. These origin sequences, however, have undergone
modifications that could be responsible for their coexistence: first,
only two of them hybridize strongly to the IncB probe and could be
amplified with primers specific for the coding sequence for the RepA
protein from pPT23A; second, the restriction pattern with
HaeIII is different among the PCR products of these two
origins and that of pAKC (Fig. 5). The nucleotide sequences of the
repA genes of pAV505, from P. syringae pv.
phaseolicola 1302A, and of pPT23A (13), two pPT23A-like
plasmids that are compatible, show ca. 89% identity. Base changes are
mainly concentrated in the DNA upstream from the start codons and in
the 3' end of the RepA coding regions. The deletion of 64 nucleotides
in this 3' end in repA from pPT23A led to an altered
incompatibility phenotype, which supports the idea that small changes
in the repA sequence would maintain a high degree of
homology within repA but also allow for coexistence.
The presence of two other strong incompatibility determinants close to
oriV-pPT23A suggests that these regions should also be
modified or absent in coexisting pPT23A-like plasmids to account for
the observed compatibility. In hybridization experiments using specific
probes for IncA, IncB, IncC, and rulAB (Fig. 2), most of the
plasmids examined hybridized only to IncB. The hybridization to IncA,
IncC, or rulAB, when present, was in all cases
associated with plasmids hybridizing to the IncB probe. Most of the
pPT23A-like replicons did not contain IncC, and in no case did we
observe hybridization to this probe in more than one plasmid of the
same cell. Since the cloned IncC determinant displays strong
incompatibility with pPT23A, its parent plasmid, it is tempting to
speculate that this determinant was lost by deletion during the
evolution of coexisting pPT23A-like replicons. Although also poorly
conserved, IncA is in some cases repeated in up to three plasmids of
the same cell. In these cases, some of the copies could no longer be
functional or might have undergone specificity changes by mutation. In
this respect, pPT23B hybridizes to IncA (Fig. 2), but the sequencing of
oriV-pPT23B (6) showed that IncA is not located
in its vicinity, as it is in oriV-pPT23A. Taking into
account that pPT23B does not hybridize to IncC or rulAB,
this suggests that the replication region in this plasmid, and probably
in other pPT23A-like plasmids, may have undergone major reorganization
events.
The existence of highly related replicons in different P. syringae strains could limit the horizontal transfer of plasmids among them. The acquisition of pAKC, which contains three
incompatibility determinants, did not result in most cases in the
eviction of any native plasmid from the 10 strains analyzed, suggesting
that the incompatibility determinants in pAKC are not functionally conserved among plasmids of the pPT23A-like family. Also, this situation could be explained in terms of partial or complete
autocompatibility, such as that observed with oriV-pPT23B
(23) and pORI602. As expected, pAKC led to the eviction from
PT23 of pPT23A, its parent plasmid. A plasmid of similar size was
also evicted from the related strains PT17 and PT30 but not from other
P. syringae pv. tomato strains. On some occasions,
pPT23B was also evicted, despite the fact that this plasmid is stably
maintained with pPT23A. It is possible that the eviction of pPT23B and
of three plasmids from P. syringae pv. glycinea race 4 was caused by a shift in the balance of repA and
ori sequences due to copy number effects of the pAKC clone.
Taken together, these data suggest that several origins of replication
of the pPT23A-like family could coexist in the same cell due to base
changes that could alter their specificity plus the incorporation,
elimination, and/or functional modification of other strong
incompatibility determinants, like IncA or IncC. For example,
each of four of the pPT23A-like plasmids from P. syringae pv. glycinea race 6 showed a differential hybridization pattern with the Inc probes (Fig. 2) but exhibited HaeIII
digestion patterns of their respective repA-PCR fragments
which were more similar to each other than to that of pAKC (Fig. 5).
This, together with the fact that they share a large amount of repeated
DNA, suggests that race 6 plasmids may have originated from a common ancestor and not as a result of horizontal transfer. However, a
detailed analysis of pPT23A-like replicons must be performed before the
possibility of horizontal transfer can be eliminated. An alternative to
explain the coexistence of pPT23A-like plasmids is that some or all of
them could contain another functional origin of replication which could
alleviate or suppress the incompatibility. We recently determined that
plasmids p485C and p485D, from P. syringae pv.
savastanoi 0485-9, which hybridized with oriV-pPT23A (Fig.
2), also hybridize with the unrelated origin of replication cloned in
pORI604 (28). This is the first evidence that some P. syringae native plasmids can indeed contain more
than one origin of replication.
The widespread occurrence of pPT23A-like plasmids among P. syringae pathovars is an indication of the evolutionary success of
this plasmid family and implies that these plasmids encode determinants
of importance to the P. syringae life cycle. The study
of the replication determinants of pPT23A-like plasmids is of
importance in (i) providing molecular tools for their eviction from the
host cell, and thus for the evaluation of the role of individual
plasmids in pathogenicity or virulence; (ii) providing insights into
the mechanisms that allow Pseudomonas strains to obtain a
greater genomic plasticity; and (iii) yielding information on plasmid
speciation and the evolution of new incompatibility groups within a
plasmid population. Several determinants involved in pathogenicity,
virulence, avirulence, resistance to antibacterial compounds or UV
light, and competitive fitness have been located to plasmids in
different P. syringae strains. The exchange of this
information is probably occurring in nature, since the conjugative transfer of plasmids among P. syringae strains has been
demonstrated in vitro as well as in planta (2, 4, 5).
Additionally, it has been shown that several native plasmids in this
species can mobilize chromosomal genes through integration and
imperfect excision. The horizontal exchange of genetic information
could be, however, hampered by the preexisting incompatibility among highly related replicons in P. syringae. Our results
show that modifications of the repA sequences and the poor
conservation of putative maintenance determinants could be
contributing to the reduction or suppression of the
incompatibility among members of the pPT23A-like family and thus
enhancing the genomic plasticity of this species.
| |
ACKNOWLEDGMENTS |
We thank D. Cooksey, N. T. Keen, and J. D. Taylor for
supplying P. syringae strains and R. Díaz-Orejas, R. Jackson, and A. Vivian for critically reading
the manuscript.
J.M. acknowledges support for this work from grants BIO94-0442 and
BIO97-0598 from the Spanish CICYT, and G.W.S. acknowledges support from
the Texas Agricultural Experiment Station.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratorio de
Patología Vegetal, Escuela Técnica Superior de Ingenieros
Agrónomos, Universidad Pública de Navarra, 31006 Pamplona,
Spain. Phone: 34-48-169133. Fax: 34-48-169169. E-mail:
jesus{at}upna.es.
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Abstract
Introduction
