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Molecular Analysis of Closely Related Copper- and Streptomycin-Resistance Plasmids inPseudomonas syringaepv. syringae
PLASMID
35, 98–107 (1996) 0012
ARTICLE NO.
Molecular Analysis of Closely Related Copper- and StreptomycinResistance Plasmids in Pseudomonas syringae pv. syringae GEORGE W. SUNDIN1
AND
CAROL L. BENDER2
110 Noble Research Center, Department of Plant Pathology, Oklahoma State University, Stillwater, Oklahoma 74078 Received August 29, 1995; revised January 12, 1996 The genetic relationship of a group of copper (Cur) and streptomycin (Smr) resistance plasmids and their Pseudomonas syringae pv. syringae hosts was examined. Each of these plasmids contained sequences homologous to the oriV and par sequences from pOSU900, a cryptic P. syringae pv. syringae plasmid. Analysis of restriction digest patterns of plasmid DNA indicated that the plasmids could be clustered into four groups; two of the groups contained multiple members which differed by only a few fragments. An analysis of the host P. syringae genotypes using the arbitrarily primed PCR technique and genomic DNA indicated that the host strains could be placed in groups similar to those resulting from analysis of plasmid DNA. Southern hybridization analyses of plasmid DNA indicated that each Smr plasmid contained sequences homologous to probes specific for the strA-strB Smr genes and the transposase and resolvase genes from Tn5393. All plasmids hybridized to two additional probes derived from P. syringae plasmid DNA, but none of the plasmids contained IS51 or IS801 sequences. Furthermore, Tn5393 was mobilized, presumably by transposition, between the incompatible plasmids pPSR5 and pPSR4 in P. syringae pv. syringae FF5. The variation in molecular structure of the closely related plasmids in this study is similar to that observed with antibiotic-resistance plasmids from clinical bacteria. q 1996 Academic Press, Inc.
The presence of indigenous plasmids in plant pathogenic bacteria is thought to confer a selective advantage to the host, although in most cases specific traits associated with the plasmids are unknown (Shaw, 1987; Coplin, 1989). The stable maintenance of plasmids in plant pathogens suggests a potential relevance to the host–pathogen interaction; indeed, the symbiotic plasmids of Rhizobium and the tumor-inducing plasmids of Agrobacterium play critical roles in the interaction of these bacteria with their respective plant hosts (Long and Staskawicz, 1993). Thus, the identification of plasmid-encoded genes and an understanding of the relationships of native plasmids and their hosts are essential in establishing the role
of these elements in the evolution of plant pathogenic bacteria. Most pathovars of Pseudomonas syringae contain indigenous plasmids, and some of these are known to be conjugative (Shaw, 1987; Coplin, 1989). Plasmid-encoded genes known to be important in the interaction of P. syringae with host plants include avirulence genes (Kobayashi et al., 1990) and genes for biosynthesis of ethylene, indoleacetic acid, and the phytotoxin coronatine (Comai and Kosuge, 1980; Bender et al., 1989; Nagahama et al., 1994). Other plasmid-encoded sequences which have been characterized from P. syringae include: (i) copper-resistance (Cur)3 determinants (Bender and Cooksey, 1987; Cooksey, 1990) and the streptomycin-resistance
1 Present address: Department of Microbiology and Immunology (M/C 790), University of Illinois College of Medicine, 835 S. Wolcott Ave., Chicago, IL 60612. 2 To whom correspondence should be addressed. Phone: 405-744-9945; Fax: 405-744-7373; e-mail: [email protected].
3 Abbreviations used: Cur, copper-resistance; Smr, streptomycin-resistance; IS, insertion sequence; Tn, transposon; MG medium, mannitol glutamate medium; AP-PCR, arbitrarily primed polymerase chain reaction; UVr, resistance to ultraviolet light; MGcu sm, MG medium containing cupric sulfate and streptomycin.
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Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Cur AND Smr PLASMIDS IN P. syringae
(Smr) transposon Tn5393 (Sundin et al., 1994); (ii) the insertion sequence (IS) elements IS51, IS52, IS53, and IS801 (Yamada et al., 1986; Romantschuk et al., 1991; Soby et al., 1993); and (iii) the origin of replication (oriV) sequences of pOSU900 and pPS10 from P. syringae pv. syringae and P. syringae pv. savastanoi, respectively (Mukhopadhyay et al., 1990; Nieto et al., 1992). Additionally, several reports have shown that the distribution of many P. syringae plasmid DNA sequences is not restricted to individual pathovars (Cooksey, 1990; Kobayashi et al., 1990; Bender et al., 1991; Romantschuk et al., 1991; Sundin and Bender, 1993; Murillo and Keen, 1994; Nagahama et al., 1994; Sundin et al., 1994; Yucel et al., 1994). We are interested in the ecology of Cur and Smr plasmids in P. syringae and are studying factors involved in the persistence of these plasmids within natural populations. We recently discovered a high level of genetic diversity among Cur and Smr strains of P. syringae pv. syringae isolated from nurseries in Oklahoma (Sundin et al., 1994). The Cur determinant and the Smr transposon Tn5393 from these strains were encoded by at least two distinct groups of plasmids (Sundin et al., 1994). One of these groups consisted of plasmid variants which contained sequences that hybridized to the oriV sequences and putative stability loci from pOSU900, a cryptic plasmid from P. syringae pv. syringae J900 (Mukhopadhyay et al., 1990). The pOSU900 replicon sequences are distributed among plasmids inhabiting many pathovars of P. syringae (Murillo and Keen, 1994; Sundin et al., 1994), implying that this plasmid group may encode other genes important to the biology of P. syringae. Our objective in the present study was to examine the genetic relationships of this group of plasmids using cluster analysis of their restriction digest patterns. Southern hybridization analyses were also used to investigate whether specific genetic determinants were conserved among these plasmids, and we also studied the potential mechanisms involved in the interplasmid dissemination of Tn5393.
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MATERIALS AND METHODS
Bacterial strains, plasmids, and media. Table 1 lists the bacterial strains and plasmids used in the present study. The P. syringae pv. syringae strains were isolated from symptomless leaves or dormant buds of ornamental pear trees (cv. Aristocrat) at three nurseries in Oklahoma. P. syringae pv. syringae was cultured at 287C on King’s medium B (King et al., 1954) or mannitol glutamate (MG) medium (Keane et al., 1970). Escherichia coli was cultured at 377C on LB medium (Miller, 1972). Cupric sulfate was added to MG medium at 250 mg/ml, and the antibiotics ampicillin and streptomycin were added to media at 40 and 25 mg/ml, respectively. Molecular genetic techniques. Restriction enzyme digests, isolation of DNA fragments from agarose gels by electroelution, molecular cloning, and Southern transfers to nylon membranes were done using published techniques (Sambrook et al., 1989). Electroporation of P. syringae pv. syringae using plasmid DNA was done using a previously described procedure (Garde and Bender, 1991). DNA fragments used as probes were labeled with digoxigenin11–dUTP (Genius kit; Boehringer-Mannheim, Indianapolis, IN) following the instructions of the manufacturer. DNA hybridizations at 657C followed by high-stringency washes were done as described previously (Sundin and Bender, 1993). Genetic analyses of plasmid and genomic DNA from P. syringae pv. syringae. Plasmids were isolated from 10-ml P. syringae pv. syringae cultures using the method of Crosa and Falkow (1981). Following isopropanol precipitation, the plasmid DNA was resuspended in TE buffer [10 mM Tris–HCl (pH 8.0), 1 mM Na2-EDTA], extracted once with phenol:chloroform:isoamyl alcohol (25:24:1) and once with chloroform:isoamyl alcohol (24:1), and precipitated with ethanol. Each plasmid was digested separately with BamHI, EcoRI, or SstI for 3 h. The digestion products were separated on 1.2% agarose gels, and restriction enzyme patterns were compared from the same gel. The restriction fragment data were converted to a two-dimensional binary matrix
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SUNDIN AND BENDER TABLE 1 BACTERIAL STRAINS Strain
Pseudomonas syringae pv. syringae 7B12 4A39 FF3 7B44 2H12 3C1 8B48 7A7 7B40 7C12 FF5 FF5(pPSR4) FF5.2 FF5.2(pPSR22) Plasmids pBluescript (SK/) pRK2013 pRK415 pGWS36 pGWS49 pOSU801b pMUBSC pOSU22 pPSR1.7 pPSR4::Tn5393a pSM1
AND
PLASMIDS
AND
Relevant characteristics
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Reference
pPSR14; Cur Smr (Tn5393) pPSR4, pPSC9, pPSC10; Cur pPSR5; Smr (Tn5393) pPSR7; Cur pPSR9; Cur pPSR10; Cur pPSR11; Cur pPSR22, pPSC11; Cur pPSR23; Cur Smr (Tn5393) pPSR13, pPSR21; Cur Smr (Tn5393) Cus Sms; no detectable plasmids pPSR4 introduced by electroporation; Cur Cmr derivative of FF5 pPSR22 introduced by conjugation; Cur
Sundin et al. (1994) This study Sundin and Bender (1993) Sundin et al. (1994) Sundin et al. (1994) Sundin et al. (1994) Sundin et al. 1994) This study This study Sundin et al. (1994) Sundin and Bender (1993) Sundin and Bender (1994) Sundin and Bender (1993) This study
Ampr, ColE1 cloning vector Kmr, Tra/, helper plasmid Tcr, broad-host-range vector 0.7-kb HindIII of pSM1 in pBluescript (SK/) 8.5-kb EcoRI of pPSR4::Tn5393a in pBluescript (SK/) Contains IS801 Contains IS51 oriV, par loci from pOSU900 Cosmid clone of pPSR1 containing Tn5393 pPSR4 evolved in vitro containing Tn5393 strA-strB Smr genes from pPSR1
Stratagene Figurski and Helinski (1979) Keen et al. (1988) G. Sundin This study Romantschuk et al. (1991) M. Ullrich Mukhopadhyay et al. (1990) Sundin and Bender (1995) This study Sundin and Bender (1993)
where 1 Å the presence of a restriction fragment and 0 Å the absence of a restriction fragment. Data analysis was performed with the biostatistical analysis program NTSYS-pc (Applied Biostatistics, Inc., Setauket, NY). A similarity matrix was computed using a simple matching coefficient, and cluster analysis was performed using the unweighted pairgroup method arithmetic average (UPGMA) (Sneath and Sokal, 1973). Southern hybridizations were conducted to determine if specific genetic determinants were located on the plasmids analyzed in this study. DNA fragments used as probes were: (i) the 1.2-kb HindIII and 1.4-kb EcoRI fragments from pOSU22 containing the oriV and par sequences, respectively, from pOSU900, a cryptic plasmid in P. syringae pv. syringae; (ii) the 1.5-kb SstI–EcoRV fragment from
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pSM1 containing the strA-strB Smr genes from Tn5393; (iii) the 3.2-kb SstI fragment from pPSR1.7 containing an internal fragment of the tnpA-tnpR genes from Tn5393; (iv) the 1.5-kb EcoRI/HindIII fragment from pOSU801b containing IS801 from P. syringae pv. phaseolicola; (v) the 1.0-kb SstI fragment from pMUBSC containing an internal fragment of IS51 from the P. syringae pv. glycinea plasmid p4180A; and (vi) the 0.7-kb HindIII fragment of pGWS36 containing an internal fragment of the ruvZY genes which confer resistance to ultraviolet light (UVr); this was obtained from P. syringae pv. syringae plasmid pPSR1 (Sundin and Bender, unpublished data). Arbitrarily primed PCR analysis. The genetic relationship between the natural P. syringae pv. syringae strains which harbored the
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plasmids analyzed in this study was examined using the arbitrarily primed polymerase chain reaction (AP-PCR) technique (Welsh and McClelland, 1990; Williams et al., 1990). An 18-bp primer, complementary to a region in IS50 from Tn5 (Rich and Willis, 1990), was used in the AP-PCR analysis. AP-PCR reactions using intact cells, data analysis, construction of similarity matrices, and cluster analysis were performed as described previously (Sundin et al., 1994). Interplasmid mobilization of Tn5393. Experiments were conducted to examine the transfer of Tn5393 between plasmids using plasmid pairs which were either compatible or incompatible. In the incompatible interaction, P. syringae pv. syringae 4A39, which contains the Cur plasmid pPSR4 and two cryptic plasmids, was mated with P. syringae pv. syringae FF3, a strain containing pPSR5, an indigenous plasmid conferring Smr due to the presence of Tn5393. These plasmids each encode homologous oriV and par sequences and were previously shown to be incompatible (Sundin and Bender, unpublished). Two independent matings were conducted and selection of FF3 containing pPSR4 and pPSR5 was on MG medium containing cupric sulfate and streptomycin (MGcu sm). The conjugation frequency of pPSR4 was approximately 103fold greater than that of pPSR5, and the results of preliminary matings indicated that the Cur Smr exconjugants selected were FF3 containing pPSR4 and pPSR5. Because pPSR4 and pPSR5 were incompatible, growth of exconjugants overnight in MG broth resulted in the rapid elimination of one of the plasmids. Therefore, separate exconjugants from each mating were incubated in batch culture under continuous copper and streptomycin selection. The bacteria were initially grown for 24 h in 5 ml MGcu sm broth; then 0.05-ml aliquots of stationary phase culture were transferred into 4.95 ml fresh MGcu sm (to maintain selection for both plasmids) and MG broth without selection (to investigate whether a single stable plasmid might evolve encoding both copper and streptomycin resistance). The 100fold daily increase in cell numbers corresponded to approximately 6.64 generations
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per day. On each succeeding day for up to 7 days, cells were subcultured from MGcu sm into MG and MGcu sm, enumerated on MG and MGcu sm agar, and examined for plasmid DNA content. In the compatible interaction, we utilized P. syringae pv. syringae 7C12, a strain containing two indigenous compatible plasmids, pPSR13, a 60-kb Cur plasmid, and pPSR21, a 200-kb Smr plasmid containing Tn5393. Strain 7C12 was grown in batch culture in the minimal medium of Hoitink and Sinden (1970) without copper or streptomycin selection and with limiting concentrations of carbon (the glucose concentration was 0.25 g/ liter). The growth limitation was imposed as an additional burden on plasmid carriage. The strain was allowed to grow approximately 6.64 generations over a 24-h period, and the experiment was continued for 100 generations. At 20-generation intervals, bacterial cells were enumerated on MG and MGcu sm media, and plasmid preparations were performed on five colonies from the MGcu sm plates. This experiment was performed twice. RESULTS
Plasmid analyses. Six Cur, one Smr, and two Cur Smr plasmids were analyzed from nine strains of P. syringae pv. syringae. Two P. syringae host strains (4A39 and 7A7) contained cryptic plasmids in addition to those mediating Cur; therefore, pPSR4 was transferred from 4A39 to the plasmidless recipient FF5 by electroporation, and pPSR22 was transferred into FF5.2 (Cmr derivative of FF5) by conjugation. The plasmids studied ranged in size from approximately 53 to 68 kb except pPSR23 which was 95 kb (data not shown). Each plasmid was previously shown to contain sequences homologous to the origin of replication and putative stability loci of pOSU900, a cryptic P. syringae pv. syringae plasmid (Mukhopadhyay et al., 1990; Sundin and Bender, 1994; Sundin et al., 1994). The pattern of restriction fragments generated with BamHI, EcoRI, and SstI were reproduced in separate plasmid preparations of the same strain over a period of months. The banding
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FIG. 1. Graphic representation of plasmid restriction fragments generated by digestion of P. syringae pv. syringae plasmid DNA with EcoRI. Plasmid designations and resistance phenotype follow the strain name in parentheses. The plasmid groups, based on cluster analysis of a similarity matrix generated from restriction fragment data, are shown above each lane. Lanes 1 to 9 contain the following strains: 1, 7B44 (pPSR7-Cur); 2, 8B48 (pPSR11-Cur); 3, FF5.2 (pPSR22-Cur); 4, 3C1 (pPSR10Cur); 5, 2H12 (pPSR9-Cur); 6, 7B12 (pPSR14-Cur Smr); 7, FF3 (pPSR5-Smr); 8, 7B40 (pPSR23-Cur Smr); 9, FF5 (pPSR4-Cur). Linear DNA size standards are indicated at the left.
patterns generated in each plasmid digest suggested that all nine plasmids were related. A diagrammatic representation of the EcoRI banding patterns is shown in Fig. 1. Fragments ranging in size from approximately 1.2 to 18 kb were scored for each plasmid/restriction enzyme combination and the data were converted into a binary matrix for quantitative comparison. Cluster analysis of the data matrix indicated that the plasmids could be assigned to four groups (A–D), with groups A and C containing multiple members which were closely related (Fig. 2a). The restriction fragment data suggested both similarities and distinctions between the plasmids, but provided no information on conserved sequences. Thus, further analyses of the plasmids consisted of Southern hybridization analyses using characterized determinants from P. syringae plasmids. DNA probes specific for the strA-strB Smr genes or the trans-
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posase and resolvase genes of Tn5393 hybridized to the Smr plasmids pPSR5, pPSR14, and pPSR23 (Table 2). Sequences homologous to the ruvZY genes cloned from pPSR1 were present on each of the plasmids (Table 2). Neither IS51 nor IS801 was detected on the plasmids examined in this study (Table 2). Arbitrarily primed PCR analysis. The APPCR technique utilizes oligonucleotide primers in low-stringency PCR reactions to generate patterns of DNA fragments which are strain-specific (Welsh and McClelland, 1990; Williams et al., 1990). Twenty-six different DNA bands, ranging in size from approximately 0.3 to 3.0 kb, were amplified from the nine strains; these bands were numbered (1 to 26) in order of descending size (Fig. 3). The number of DNA bands amplified per strain ranged from two to fifteen (Fig. 3). The data indicated that many bands were detected in more than one strain and that the general pat-
FIG. 2. (a) Dendrogram showing relatedness of nine P. syringae pv. syringae plasmids. The dendrogram was generated from similarity matrices of restriction fragment patterns. Plasmid group designations are shown at the right. (b) Dendrogram of nine P. syringae pv. syringae strains analyzed by the arbitrarily primed polymerase chain reaction technique (AP-PCR). A similarity matrix derived from the DNA banding patterns obtained by APPCR and the IS50 primer was used to generate the dendrogram. Resident plasmids in P. syringae pv. syringae are shown at the right.
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Cur AND Smr PLASMIDS IN P. syringae TABLE 2 DETECTION
OF
SPECIFIC SEQUENCES
IN
P. syringae pv. syringae PLASMIDS Hybridization toa
Plasmid
Groupb
oriV and par
strA-strB
tnpA-tnpR
ruvZY
IS801
IS51
pPSR14 pPSR4 pPSR5 pPSR7 pPSR9 pPSR10 pPSR11 pPSR22 pPSR23
C B C A C A A A D
// // // // // // // // //
// 00 // 00 00 00 00 00 //
// 00 // 00 00 00 00 00 //
// // // // // // // // //
00 00 00 00 00 00 00 00 00
00 00 00 00 00 00 00 00 00
a //, indicates hybridization was observed; 00, indicates no hybridization was observed. The sequences used as probes were: the oriV and par sequences from pOSU900; these were used separately but produced identical results; strA-strB Smr genes and the tnpA and tnpR genes from Tn5393; ruvZY, a determinant from pPSR1 which confers resistance to ultraviolet light; and IS801 and IS51, insertion sequences from P. syringae. b The plasmid groups were assigned based on restriction fragment pattern data (see Fig. 3A).
tern of bands was conserved among groups of strains (Fig. 3). For example, strains 7B44 and 7A7 contain group A plasmids and produced identical banding patterns when their genomic DNA was analyzed by AP-PCR. A data matrix was generated utilizing each strain and scoring for the presence or absence of a particular DNA band. Cluster analysis of the AP-PCR data matrix indicated that all of the P. syringae pv. syringae host strains were clustered into groups which were reflective of the plasmid groups (Fig. 2b).
Interplasmid mobilization of Tn5393. The potential for mobilization of Tn5393 among P. syringae pv. syringae plasmids was assessed using both a compatible and an incompatible plasmid pair. In the incompatible interaction, we monitored for the evolution of a single Cur Smr plasmid following the generation of a strain carrying the two incompatible plasmids, pPSR4 (Cur) and pPSR5 (Smr::Tn5393). The results of two independent matings indicated that both pPSR4 and pPSR5 were initially maintained when the
FIG. 3. Matrix of DNA banding patterns observed for P. syringae pv. syringae strains using the arbitrarily primed polymerase chain reaction technique and the IS50 primer. The DNA bands were numbered from 1 to 26 in order of descending size. ‘‘X’’ indicates the presence of a particular band, and a blank space indicates the absence of that band. The sizes of the amplified DNA fragments are as follows: 1, 3.0 kb; 2, 2.6 kb; 3, 2.5 kb; 4, 2.1 kb; 5, 1.9 kb; 6, 1.5 kb; 7, 1.4 kb; 8, 1.3 kb; 9, 1.25 kb; 10, 1.2 kb; 11, 1.15 kb; 12, 1.1 kb; 13, 1.05 kb; 14, 1.0 kb; 15, 0.95 kb; 16, 0.9 kb; 17, 0.85 kb; 18, 0.8 kb; 19, 0.7 kb; 20, 0.65 kb; 21, 0.55 kb; 22, 0.5 kb; 23, 0.45 kb; 24, 0.4 kb; 25, 0.35 kb; and 26, 0.3 kb.
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FIG. 4. (A) Movement of Tn5393 between two incompatible plasmids in P. syringae pv. syringae FF3. Plasmid designations follow the strain name in parentheses. Lanes 1 to 4 contain the following strains: 1, FF5 (pPSR4); 2, FF3 (pPSR5); 3, FF3 (pPSR4 and pPSR5); 4, FF3 (pPSR4::Tn5393). To simplify presentation of the results, FF5(pPSR4) is shown in lane 1 instead of the actual donor strain 4A39 since the latter contains two cryptic plasmids in addition to pPSR4. Molecular weights (kb) are indicated at the left. (B) Restriction fragment analysis of selected plasmids. Lanes 1 to 3 contain EcoRI digestion products of the following plasmids: 1, pPSR5; 2, pPSR4; 3, pPSR4::Tn5393. Arrow at right indicates EcoRI fragment containing Tn5393. Linear DNA size standards are indicated at the left.
cells were grown with copper and streptomycin selection (Fig. 4A, lane 3). By subculturing the cells in both MGcu sm and MG, we could track the evolution of a single plasmid encoding both copper and streptomycin resistance by correlation with the stability of both determinants in MG broth lacking Cu and Sm. Following a growth period of 32 { 8 generations in MGcu sm broth, Tn5393 was mobilized, presumably by transposition, from pPSR5 to pPSR4; this event was associated with the loss of pPSR5 from the cells (Fig. 4A, lane 4). Analysis of the resulting Cur Smr plasmids by digestion with EcoRI (there are no sites for EcoRI within Tn5393) and hybridization to the strA-strB and tnpA-tnpR probes, indicated that Tn5393 was present on the evolved pPSR4 plasmid in all cases and that it was present in the same 8.5-kb EcoRI restriction fragment (Fig. 4B, lane 3, see arrow). This was correlated with the disappearance of a faintly visible 3.0-kb EcoRI fragment in pPSR4 (Fig. 4B, lane 2, arrow), an event which is consistent with the insertion of the 5.5-kb Tn5393 into this fragment (Sundin and
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Bender, 1995). The 8.5-kb fragment was also cloned into pBluescript (SK/) to generate the recombinant pGWS49; this clone conferred Smr to E. coli and was used to confirm the presence of Tn5393 within the 8.5-kb fragment. In the compatible plasmid interaction, the wild-type P. syringae pv. syringae strain 7C12, which contains two compatible plasmids, pPSR13 (Cur) and pPSR21 (Smr due to Tn5393) was grown for 100 generations in a minimal medium under limited glucose to determine if Tn5393 would move from the large Smr plasmid to the smaller Cur plasmid or to the chromosome. In two separate experiments, there were no observable changes in the size of either plasmid following 100 generations of growth (data not shown). DISCUSSION
In this study, we examined the relatedness of Cur and Smr plasmids sharing a common replicon and their P. syringae pv. syringae hosts. Cluster analysis of the restriction digest patterns indicated that the plasmids could be organized into four groups. Two groups (A and C) contained multiple members which differed only slightly in restriction fragment digest patterns. The conservation of restriction fragments, DNA sequences, and phenotypic traits among the plasmids in these groups suggests that they comprise stable ‘‘cohesive lineages’’ as defined by Riley and Gordon for ColE1 plasmids (Riley and Gordon, 1992). A comparison of the relationships between plasmids and their P. syringae pv. syringae hosts indicated that their phylogenetic trees are quite similar (Figs. 2a and 2b). These results suggest that no recent plasmid transfer events have occurred. The similar phylogenetic trees obtained for the plasmids and their P. syringae hosts and genetic distance between some plasmids (e.g., pPSR7 and pPSR23) and their host strains (7B44 and 7B40, respectively) implies that these plasmids have resided in their hosts for long periods of time. Our previous population study indicated that epiphytic P. syringae pv. syringae strains from ornamental pear could be
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differentiated into two divergent groups (Sundin et al., 1994). Interestingly, specific plasmid profiles were confined to P. syringae pv. syringae host genotypes from a single group (Sundin et al., 1994). Results obtained in the current study confirm these data and imply that the P. syringae pv. syringae plasmids we have studied may be disseminated among groups of related strains but not among more distantly related strains. The compartmentalization of plasmids within specific host genotypes has also been observed in Rhizobium leguminosarum (Young and Wexler, 1988). Epidemiological studies of antibiotic-resistance plasmids have shown that subpopulations of plasmid variants commonly occur within host bacterial populations (e.g., Hopkins et al., 1986; Tran van Hieu et al., 1986; Platt et al., 1988). These variants may contain subtle differences in restriction digest pattern or obvious additions, deletions, or rearrangements of DNA. In many cases, the evolution of multiple antibiotic-resistance plasmids is thought to have occurred via the sequential insertion of transposable elements (LabigneRoussel et al., 1982; Saunders et al., 1986). In this study, the plasmids in individual groups appear to differ only by minor alterations in restriction digest profiles (Fig. 1). In addition, the Cur Smr plasmid pPSR14 may have evolved from the Cur plasmid pPSR9 through the insertion of Tn5393 in a 5.1-kb EcoRI fragment (Fig. 1; lanes 5–6). Thus, the P. syringae pv. syringae plasmids examined in this study may have evolved via mechanisms observed in antibiotic-resistance plasmids inhabiting clinically important bacteria. The plasmid evolution experiments reported here showed that pPSR4 could acquire Tn5393 in vitro. Theoretical and experimental considerations which could promote the evolution of a single multiresistance plasmid from incompatible plasmids have been reported for E. coli (Condit and Levin, 1990). Like Condit and Levin (1990), we did not detect movement of the transposon between compatible plasmids located in the same host cell. The movement of Tn5393 between plasmids is presumably responsible for the presence of the element on plasmids of a variety of sizes within
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natural P. syringae populations (Jones et al., 1991; Sundin et al., 1994). The interplasmid mobilization of Tn5393 mediated by plasmid incompatibility may commonly occur in natural populations; conjugative plasmids containing homologous oriV and par sequences with the plasmid group in this study are common in P. syringae pv. syringae populations (Sundin et al., 1994). Alternatively, Tn5393 could be acquired by a cell from a suicide plasmid, i.e., a Tn5393-containing plasmid which could conjugate to P. syringae but not replicate within the cell. Tn5393 is commonly harbored on large plasmids in plant and soil bacterial populations which co-inhabit nurseries with P. syringae pv. syringae populations (Sundin et al., 1995). Our results indicate that IS51 and IS801, which are associated with plasmid-encoded virulence determinants in other P. syringae pathovars (Soby et al., 1994; Ullrich and Bender, unpublished observations), have not colonized the P. syringae pv. syringae plasmids characterized in the present study. Both IS51 and IS801 have colonized plasmids containing the pOSU900 replicon in other P. syringae pathovars (Murillo and Keen, 1994; Ullrich and Bender, unpublished observations). While plasmids containing the pOSU900 replicon have been detected in at least 16 pathovars (Murillo and Keen, 1994; Sundin et al., 1994), some of the genetic determinants associated with these plasmids are more restricted in distribution. It has been suggested that the conservation of P. syringae plasmids among strains reflects a selective advantage conferred to the P. syringae host (Shaw, 1987). However, many sequences conserved among the P. syringae plasmids have no obvious relationship to pathogenesis (Obukowicz and Shaw, 1985). The UVr determinant conserved among the plasmids in the present study may be important in the survival and propagation of the P. syringae host during the epiphytic phase of its life cycle. Our recent field and growth chamber studies demonstrated that pPSR1, pPSR4, pPSR4::Tn5393a, and pPSR5 did not reduce the epiphytic fitness of P. syringae pv. syringae FF5 in the phylloplane of bean or
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ornamental pear plants (Sundin and Bender, 1994). It is possible that this group and other groups of conserved P. syringae plasmids contain a ‘‘backbone’’ of genes important for ecological fitness in the phylloplane. These plasmids may also become targets into which Cur and Smr genes are inserted. Since not all P. syringae pv. syringae strains contain indigenous plasmids, it would be worthwhile to determine the impact of conserved plasmid-encoded genes on the colonization of particular environmental niches by isogenic plasmidcontaining and plasmid-free strains. ACKNOWLEDGMENTS This research was supported by the Oklahoma Agricultural Experiment Station and National Science Foundation Grant EHR-9108771.
REFERENCES BENDER, C. L., AND COOKSEY, D. A. (1987). Molecular cloning of copper resistance genes from Pseudomonas syringae pv. tomato. J. Bacteriol. 169, 470–474. BENDER, C. L., MALVICK, D. K., AND MITCHELL, R. E. (1989). Plasmid-mediated production of the phytotoxin coronatine in Pseudomonas syringae pv. tomato. J. Bacteriol. 171, 807–812. BENDER, C. L., YOUNG, S. A., AND MITCHELL, R. E. (1991). Conservation of plasmid DNA sequences in coronatine-producing pathovars of Pseudomonas syringae. Appl. Environ. Microbiol. 57, 993–999. COMAI, L., AND KOSUGE, T. (1980). Involvement of plasmid deoxyribonucleic acid in indoleacetic acid synthesis in Pseudomonas savastanoi. J. Bacteriol. 143, 950– 957. CONDIT, R., AND LEVIN, B. R. (1990). The evolution of plasmids carrying multiple resistance genes: The role of segregation, transposition, and homologous recombination. Am. Nat. 135, 573–596. COOKSEY, D. A. (1990). Plasmid-determined copper resistance in Pseudomonas syringae from impatiens. Appl. Environ. Microbiol. 56, 13–16. COPLIN, D. L. (1989). Plasmids and their role in the evolution of plant pathogenic bacteria. Annu. Rev. Phytopathol. 27, 187–212. CROSA, J. H., AND FALKOW, S. (1981). Plasmids. In ‘‘Manual of Methods for General Bacteriology’’ (P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg, and G. B. Phillips, Eds.), pp. 266–282. Am. Soc. Microbiol., Washington, DC. FIGURSKI, D. H., AND HELINSKI, D. R. (1979). Replication of an origin containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acad. Sci. USA 79, 1648–1652.
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GARDE, S., AND BENDER, C. L. (1991). DNA probes for detection of copper resistance genes in Xanthomonas campestris pv. vesicatoria. Appl. Environ. Microbiol. 57, 2435–2439. HOITINK, H. A. J., AND SINDEN, S. L. (1970). Partial purification and properties of chlorosis-inducing toxins of Pseudomonas phaseolicola and Pseudomonas glycinea. Phytopathology 60, 1236–1237. HOPKINS, J. D., MAYER, K. H., GILLEECE, E. S., O’BRIEN, T. F., AND SYVANEN, M. (1986). Genetic and physical characterization of IncM plasmid pBWH1 and its variance among natural isolates. J. Bacteriol. 165, 47–52. JONES, A. L., NORELLI, J. L., AND EHRET, G. R. (1991). Detection of streptomycin-resistant Pseudomonas syringae pv. papulans in Michigan apple orchards. Plant Dis. 75, 529–531. KEANE, P. J., KERR, A., AND NEW, P. B. (1970). Crown gall of stone fruit. II. Identification and nomenclature of Agrobacterium isolates. Aust. J. Biol. Sci. 23, 585– 595. KEEN, N. T., TAMAKI, S., KOBAYASHI, D., AND TROLLINGER, D. (1988). Improved broad host-range plasmids for cloning in gram-negative bacteria. Gene 70, 191– 197. KING, E. O., WARD, N. K., AND RANEY, D. E. (1954). Two simple media for the demonstration of pyocyanin and fluorescein. J. Lab. Clin. Med. 44, 301–307. KOBAYASHI, D. Y., TAMAKI, S. J., AND KEEN, N. T. (1990). Molecular characterization of avirulence gene D from Pseudomonas syringae pv. tomato. Mol. Plant– Microbe Interact. 3, 94–102. LABIGNE-ROUSSEL, A., WITCHITZ, J., AND COURVALIN, P. (1982). Modular evolution of disseminated Inc 7-M plasmids encoding gentamicin resistance. Plasmid 8, 215–231. LONG, S. R., AND STASKAWICZ, B. J. (1993). Prokaryotic plant parasites. Cell 73, 921–935. MILLER, J. H. (1972). ‘‘Experiments in Molecular Genetics.’’ Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. MUKHOPADHYAY, P., MUKHOPADHYAY, M., AND MILLS, D. (1990). Construction of a stable shuttle vector for high-frequency transformation in Pseudomonas syringae pv. syringae. J. Bacteriol. 172, 477–480. MURILLO, J., AND KEEN, N. T. (1994). Two native plasmids of Pseudomonas syringae pathovar tomato strain PT23 share a large amount of repeated DNA, including replication sequences. Mol. Microbiol. 12, 941–950. NAGAHAMA, K., YOSHINO, K., MATSUOKA, M., SATO, M., TANASE, S., OGAWA, T., AND FUKUDA, H. (1994). Ethylene production by strains of the plant-pathogenic bacterium Pseudomonas syringae depends upon the presence of indigenous plasmids carrying homologous genes for the ethylene-forming enzyme. Microbiology 140, 2309–2313. NIETO, C., GIRALDO, R., FERNANDEZ-TRESGUERRES, E., AND DIAZ, R. (1992). Genetic and functional analysis of the basic replicon of pPS10, a plasmid specific for Pseudomonas isolated from Pseudomonas syringae pathovar savastanoi. J. Mol. Biol. 223, 415–426.
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Cur AND Smr PLASMIDS IN P. syringae OBUKOWICZ, M., AND SHAW, P. D. (1985). Construction of Tn3-containing plasmids from plant-pathogenic pseudomonads and an examination of their biological properties. Appl. Environ. Microbiol. 49, 468–473. PLATT, D. J., TAGGART, J., AND HERAGHTY, K. A. (1988). Molecular divergence of the serotype-specific plasmid (pSLT) among strains of Salmonella typhimurium of human and veterinary origin and comparison of pSLT with the serotype specific plasmids of S. enteritidis and S. dublin. J. Med. Microbiol. 27, 277–284. RICH, J. J., AND WILLIS, D. K. (1990). A single oligonucleotide probe can be used to rapidly isolate DNA sequences flanking a transposon Tn5 insertion by the polymerase chain reaction. Nucleic Acids Res. 18, 6673–6676. RILEY, M. A., AND GORDON, D. M. (1992). A survey of Col plasmids in natural isolates of Escherichia coli and an investigation into the stability of Col-plasmid lineages. J. Gen. Microbiol. 138, 1345–1352. ROMANTSCHUK, M., RICHTER, G. Y., MUKHOPADHYAY, P., AND MILLS, D. (1991). IS801, an insertion sequence element isolated from Pseudomonas syringae pathovar phaseolicola. Mol. Microbiol. 5, 617–622. SAMBROOK, J., FRITSCH, E. F., AND MANIATIS, T. (1989). ‘‘Molecular Cloning: A Laboratory Manual.’’ Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. SAUNDERS, J. R., HART, C. A., AND SAUNDERS, V. A. (1986). Plasmid-mediated resistance to b-lactam antibiotics in Gram-negative bacteria: The role of in-vivo recyclization reactions in plasmid evolution. J. Antimicrob. Chemother. 18(Suppl. C), 57–66. SHAW, P. D. (1987). Plasmid ecology. In ‘‘Plant Microbe Interactions’’ (T. Kosuge and E. Nester, Eds.), Vol. 2, pp. 3–39. Macmillan Co., New York. SNEATH, P. H. A., AND SOKAL, R. R. (1973). ‘‘Numerical Taxonomy.’’ Freeman, San Francisco. SOBY, S., KIRKPATRICK, B., AND KOSUGE, T. (1993). Characterization of an insertion sequence (IS53) located within IS51 on the iaa-containing plasmid of Pseudomonas syringae pv. savastanoi. Plasmid 29, 135–141. SOBY, S., KIRKPATRICK, B., AND KOSUGE, T. (1994). Characterization of high-frequency deletions in the iaacontaining plasmid, pIAA2, of Pseudomonas syringae pv. savastanoi. Plasmid 31, 21–30. SUNDIN, G. W., AND BENDER, C. L. (1993). Ecological
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and genetic analysis of copper and streptomycin resistance in Pseudomonas syringae pv. syringae. Appl. Environ. Microbiol. 59, 1018–1024. SUNDIN, G. W., AND BENDER, C. L. (1994). Relative fitness in vitro and in planta of Pseudomonas syringae strains containing copper and streptomycin resistance plasmids. Can. J. Microbiol. 40, 279–285. SUNDIN, G. W., AND BENDER, C. L. (1995). Expression of the strA-strB streptomycin resistance genes in Pseudomonas syringae and Xanthomonas campestris and characterization of IS6100 in X. campestris. Appl. Environ. Microbiol. 61, 2891–2897. SUNDIN, G. W., DEMEZAS, D. H., AND BENDER, C. L. (1994). Plasmid and genetic diversity of Pseudomonas syringae isolates differing in exposure to copper and streptomycin bactericides. Appl. Environ. Microbiol. 60, 4421–4431. SUNDIN, G. W., MONKS, D. E., AND BENDER, C. L. (1995). Distribution of the streptomycin-resistance transposon Tn5393 among phylloplane and soil bacteria from managed agricultural habitats. Can. J. Microbiol. 41, 792– 799. TRAN VAN NHIEU, G., GOLDSTEIN, F. W., PINTO, M. E., ACAR, J. F., AND COLLATZ, E. (1986). Transfer of amikacin resistance by closely related plasmids in members of the family Enterobacteriaceae isolated in Chile. Antimicrob. Agents Chemother. 29, 833–837. WELSH, J., AND MCCLELLAND, M. (1990). Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18, 7213–7218. WILLIAMS, J. G. K., KUBELIK, A. R., LIVAK, K. J., RAFALSKI, J. A., AND TINGEY, S. V. (1990). DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18, 6531–6535. YAMADA, T., LEE, P.-D., AND KOSUGE, T. (1986). Insertion sequence elements of Pseudomonas savastanoi: nucleotide sequence and homology with Agrobacterium tumefaciens transfer DNA. Proc. Natl. Acad. Sci. USA 83, 8263–8267. YOUNG, J. P. W., AND WEXLER, M. (1988). Sym plasmid and chromosomal genotypes are correlated in field populations of Rhizobium leguminosarum. J. Gen. Microbiol. 134, 2731–2739. YUCEL, I., BOYD, C., DEBNAM, Q., AND KEEN, N. T. (1994). Two different classes of avrD alleles occur in pathovars of Pseudomonas syringae. Mol. Plant–Microbe Interact. 7, 131–139. Communicated by S. B. Levy
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ARTICLE NO.
Molecular Analysis of Closely Related Copper- and StreptomycinResistance Plasmids in Pseudomonas syringae pv. syringae GEORGE W. SUNDIN1
AND
CAROL L. BENDER2
110 Noble Research Center, Department of Plant Pathology, Oklahoma State University, Stillwater, Oklahoma 74078 Received August 29, 1995; revised January 12, 1996 The genetic relationship of a group of copper (Cur) and streptomycin (Smr) resistance plasmids and their Pseudomonas syringae pv. syringae hosts was examined. Each of these plasmids contained sequences homologous to the oriV and par sequences from pOSU900, a cryptic P. syringae pv. syringae plasmid. Analysis of restriction digest patterns of plasmid DNA indicated that the plasmids could be clustered into four groups; two of the groups contained multiple members which differed by only a few fragments. An analysis of the host P. syringae genotypes using the arbitrarily primed PCR technique and genomic DNA indicated that the host strains could be placed in groups similar to those resulting from analysis of plasmid DNA. Southern hybridization analyses of plasmid DNA indicated that each Smr plasmid contained sequences homologous to probes specific for the strA-strB Smr genes and the transposase and resolvase genes from Tn5393. All plasmids hybridized to two additional probes derived from P. syringae plasmid DNA, but none of the plasmids contained IS51 or IS801 sequences. Furthermore, Tn5393 was mobilized, presumably by transposition, between the incompatible plasmids pPSR5 and pPSR4 in P. syringae pv. syringae FF5. The variation in molecular structure of the closely related plasmids in this study is similar to that observed with antibiotic-resistance plasmids from clinical bacteria. q 1996 Academic Press, Inc.
The presence of indigenous plasmids in plant pathogenic bacteria is thought to confer a selective advantage to the host, although in most cases specific traits associated with the plasmids are unknown (Shaw, 1987; Coplin, 1989). The stable maintenance of plasmids in plant pathogens suggests a potential relevance to the host–pathogen interaction; indeed, the symbiotic plasmids of Rhizobium and the tumor-inducing plasmids of Agrobacterium play critical roles in the interaction of these bacteria with their respective plant hosts (Long and Staskawicz, 1993). Thus, the identification of plasmid-encoded genes and an understanding of the relationships of native plasmids and their hosts are essential in establishing the role
of these elements in the evolution of plant pathogenic bacteria. Most pathovars of Pseudomonas syringae contain indigenous plasmids, and some of these are known to be conjugative (Shaw, 1987; Coplin, 1989). Plasmid-encoded genes known to be important in the interaction of P. syringae with host plants include avirulence genes (Kobayashi et al., 1990) and genes for biosynthesis of ethylene, indoleacetic acid, and the phytotoxin coronatine (Comai and Kosuge, 1980; Bender et al., 1989; Nagahama et al., 1994). Other plasmid-encoded sequences which have been characterized from P. syringae include: (i) copper-resistance (Cur)3 determinants (Bender and Cooksey, 1987; Cooksey, 1990) and the streptomycin-resistance
1 Present address: Department of Microbiology and Immunology (M/C 790), University of Illinois College of Medicine, 835 S. Wolcott Ave., Chicago, IL 60612. 2 To whom correspondence should be addressed. Phone: 405-744-9945; Fax: 405-744-7373; e-mail: [email protected].
3 Abbreviations used: Cur, copper-resistance; Smr, streptomycin-resistance; IS, insertion sequence; Tn, transposon; MG medium, mannitol glutamate medium; AP-PCR, arbitrarily primed polymerase chain reaction; UVr, resistance to ultraviolet light; MGcu sm, MG medium containing cupric sulfate and streptomycin.
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Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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(Smr) transposon Tn5393 (Sundin et al., 1994); (ii) the insertion sequence (IS) elements IS51, IS52, IS53, and IS801 (Yamada et al., 1986; Romantschuk et al., 1991; Soby et al., 1993); and (iii) the origin of replication (oriV) sequences of pOSU900 and pPS10 from P. syringae pv. syringae and P. syringae pv. savastanoi, respectively (Mukhopadhyay et al., 1990; Nieto et al., 1992). Additionally, several reports have shown that the distribution of many P. syringae plasmid DNA sequences is not restricted to individual pathovars (Cooksey, 1990; Kobayashi et al., 1990; Bender et al., 1991; Romantschuk et al., 1991; Sundin and Bender, 1993; Murillo and Keen, 1994; Nagahama et al., 1994; Sundin et al., 1994; Yucel et al., 1994). We are interested in the ecology of Cur and Smr plasmids in P. syringae and are studying factors involved in the persistence of these plasmids within natural populations. We recently discovered a high level of genetic diversity among Cur and Smr strains of P. syringae pv. syringae isolated from nurseries in Oklahoma (Sundin et al., 1994). The Cur determinant and the Smr transposon Tn5393 from these strains were encoded by at least two distinct groups of plasmids (Sundin et al., 1994). One of these groups consisted of plasmid variants which contained sequences that hybridized to the oriV sequences and putative stability loci from pOSU900, a cryptic plasmid from P. syringae pv. syringae J900 (Mukhopadhyay et al., 1990). The pOSU900 replicon sequences are distributed among plasmids inhabiting many pathovars of P. syringae (Murillo and Keen, 1994; Sundin et al., 1994), implying that this plasmid group may encode other genes important to the biology of P. syringae. Our objective in the present study was to examine the genetic relationships of this group of plasmids using cluster analysis of their restriction digest patterns. Southern hybridization analyses were also used to investigate whether specific genetic determinants were conserved among these plasmids, and we also studied the potential mechanisms involved in the interplasmid dissemination of Tn5393.
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MATERIALS AND METHODS
Bacterial strains, plasmids, and media. Table 1 lists the bacterial strains and plasmids used in the present study. The P. syringae pv. syringae strains were isolated from symptomless leaves or dormant buds of ornamental pear trees (cv. Aristocrat) at three nurseries in Oklahoma. P. syringae pv. syringae was cultured at 287C on King’s medium B (King et al., 1954) or mannitol glutamate (MG) medium (Keane et al., 1970). Escherichia coli was cultured at 377C on LB medium (Miller, 1972). Cupric sulfate was added to MG medium at 250 mg/ml, and the antibiotics ampicillin and streptomycin were added to media at 40 and 25 mg/ml, respectively. Molecular genetic techniques. Restriction enzyme digests, isolation of DNA fragments from agarose gels by electroelution, molecular cloning, and Southern transfers to nylon membranes were done using published techniques (Sambrook et al., 1989). Electroporation of P. syringae pv. syringae using plasmid DNA was done using a previously described procedure (Garde and Bender, 1991). DNA fragments used as probes were labeled with digoxigenin11–dUTP (Genius kit; Boehringer-Mannheim, Indianapolis, IN) following the instructions of the manufacturer. DNA hybridizations at 657C followed by high-stringency washes were done as described previously (Sundin and Bender, 1993). Genetic analyses of plasmid and genomic DNA from P. syringae pv. syringae. Plasmids were isolated from 10-ml P. syringae pv. syringae cultures using the method of Crosa and Falkow (1981). Following isopropanol precipitation, the plasmid DNA was resuspended in TE buffer [10 mM Tris–HCl (pH 8.0), 1 mM Na2-EDTA], extracted once with phenol:chloroform:isoamyl alcohol (25:24:1) and once with chloroform:isoamyl alcohol (24:1), and precipitated with ethanol. Each plasmid was digested separately with BamHI, EcoRI, or SstI for 3 h. The digestion products were separated on 1.2% agarose gels, and restriction enzyme patterns were compared from the same gel. The restriction fragment data were converted to a two-dimensional binary matrix
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Pseudomonas syringae pv. syringae 7B12 4A39 FF3 7B44 2H12 3C1 8B48 7A7 7B40 7C12 FF5 FF5(pPSR4) FF5.2 FF5.2(pPSR22) Plasmids pBluescript (SK/) pRK2013 pRK415 pGWS36 pGWS49 pOSU801b pMUBSC pOSU22 pPSR1.7 pPSR4::Tn5393a pSM1
AND
PLASMIDS
AND
Relevant characteristics
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Reference
pPSR14; Cur Smr (Tn5393) pPSR4, pPSC9, pPSC10; Cur pPSR5; Smr (Tn5393) pPSR7; Cur pPSR9; Cur pPSR10; Cur pPSR11; Cur pPSR22, pPSC11; Cur pPSR23; Cur Smr (Tn5393) pPSR13, pPSR21; Cur Smr (Tn5393) Cus Sms; no detectable plasmids pPSR4 introduced by electroporation; Cur Cmr derivative of FF5 pPSR22 introduced by conjugation; Cur
Sundin et al. (1994) This study Sundin and Bender (1993) Sundin et al. (1994) Sundin et al. (1994) Sundin et al. (1994) Sundin et al. 1994) This study This study Sundin et al. (1994) Sundin and Bender (1993) Sundin and Bender (1994) Sundin and Bender (1993) This study
Ampr, ColE1 cloning vector Kmr, Tra/, helper plasmid Tcr, broad-host-range vector 0.7-kb HindIII of pSM1 in pBluescript (SK/) 8.5-kb EcoRI of pPSR4::Tn5393a in pBluescript (SK/) Contains IS801 Contains IS51 oriV, par loci from pOSU900 Cosmid clone of pPSR1 containing Tn5393 pPSR4 evolved in vitro containing Tn5393 strA-strB Smr genes from pPSR1
Stratagene Figurski and Helinski (1979) Keen et al. (1988) G. Sundin This study Romantschuk et al. (1991) M. Ullrich Mukhopadhyay et al. (1990) Sundin and Bender (1995) This study Sundin and Bender (1993)
where 1 Å the presence of a restriction fragment and 0 Å the absence of a restriction fragment. Data analysis was performed with the biostatistical analysis program NTSYS-pc (Applied Biostatistics, Inc., Setauket, NY). A similarity matrix was computed using a simple matching coefficient, and cluster analysis was performed using the unweighted pairgroup method arithmetic average (UPGMA) (Sneath and Sokal, 1973). Southern hybridizations were conducted to determine if specific genetic determinants were located on the plasmids analyzed in this study. DNA fragments used as probes were: (i) the 1.2-kb HindIII and 1.4-kb EcoRI fragments from pOSU22 containing the oriV and par sequences, respectively, from pOSU900, a cryptic plasmid in P. syringae pv. syringae; (ii) the 1.5-kb SstI–EcoRV fragment from
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pSM1 containing the strA-strB Smr genes from Tn5393; (iii) the 3.2-kb SstI fragment from pPSR1.7 containing an internal fragment of the tnpA-tnpR genes from Tn5393; (iv) the 1.5-kb EcoRI/HindIII fragment from pOSU801b containing IS801 from P. syringae pv. phaseolicola; (v) the 1.0-kb SstI fragment from pMUBSC containing an internal fragment of IS51 from the P. syringae pv. glycinea plasmid p4180A; and (vi) the 0.7-kb HindIII fragment of pGWS36 containing an internal fragment of the ruvZY genes which confer resistance to ultraviolet light (UVr); this was obtained from P. syringae pv. syringae plasmid pPSR1 (Sundin and Bender, unpublished data). Arbitrarily primed PCR analysis. The genetic relationship between the natural P. syringae pv. syringae strains which harbored the
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plasmids analyzed in this study was examined using the arbitrarily primed polymerase chain reaction (AP-PCR) technique (Welsh and McClelland, 1990; Williams et al., 1990). An 18-bp primer, complementary to a region in IS50 from Tn5 (Rich and Willis, 1990), was used in the AP-PCR analysis. AP-PCR reactions using intact cells, data analysis, construction of similarity matrices, and cluster analysis were performed as described previously (Sundin et al., 1994). Interplasmid mobilization of Tn5393. Experiments were conducted to examine the transfer of Tn5393 between plasmids using plasmid pairs which were either compatible or incompatible. In the incompatible interaction, P. syringae pv. syringae 4A39, which contains the Cur plasmid pPSR4 and two cryptic plasmids, was mated with P. syringae pv. syringae FF3, a strain containing pPSR5, an indigenous plasmid conferring Smr due to the presence of Tn5393. These plasmids each encode homologous oriV and par sequences and were previously shown to be incompatible (Sundin and Bender, unpublished). Two independent matings were conducted and selection of FF3 containing pPSR4 and pPSR5 was on MG medium containing cupric sulfate and streptomycin (MGcu sm). The conjugation frequency of pPSR4 was approximately 103fold greater than that of pPSR5, and the results of preliminary matings indicated that the Cur Smr exconjugants selected were FF3 containing pPSR4 and pPSR5. Because pPSR4 and pPSR5 were incompatible, growth of exconjugants overnight in MG broth resulted in the rapid elimination of one of the plasmids. Therefore, separate exconjugants from each mating were incubated in batch culture under continuous copper and streptomycin selection. The bacteria were initially grown for 24 h in 5 ml MGcu sm broth; then 0.05-ml aliquots of stationary phase culture were transferred into 4.95 ml fresh MGcu sm (to maintain selection for both plasmids) and MG broth without selection (to investigate whether a single stable plasmid might evolve encoding both copper and streptomycin resistance). The 100fold daily increase in cell numbers corresponded to approximately 6.64 generations
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per day. On each succeeding day for up to 7 days, cells were subcultured from MGcu sm into MG and MGcu sm, enumerated on MG and MGcu sm agar, and examined for plasmid DNA content. In the compatible interaction, we utilized P. syringae pv. syringae 7C12, a strain containing two indigenous compatible plasmids, pPSR13, a 60-kb Cur plasmid, and pPSR21, a 200-kb Smr plasmid containing Tn5393. Strain 7C12 was grown in batch culture in the minimal medium of Hoitink and Sinden (1970) without copper or streptomycin selection and with limiting concentrations of carbon (the glucose concentration was 0.25 g/ liter). The growth limitation was imposed as an additional burden on plasmid carriage. The strain was allowed to grow approximately 6.64 generations over a 24-h period, and the experiment was continued for 100 generations. At 20-generation intervals, bacterial cells were enumerated on MG and MGcu sm media, and plasmid preparations were performed on five colonies from the MGcu sm plates. This experiment was performed twice. RESULTS
Plasmid analyses. Six Cur, one Smr, and two Cur Smr plasmids were analyzed from nine strains of P. syringae pv. syringae. Two P. syringae host strains (4A39 and 7A7) contained cryptic plasmids in addition to those mediating Cur; therefore, pPSR4 was transferred from 4A39 to the plasmidless recipient FF5 by electroporation, and pPSR22 was transferred into FF5.2 (Cmr derivative of FF5) by conjugation. The plasmids studied ranged in size from approximately 53 to 68 kb except pPSR23 which was 95 kb (data not shown). Each plasmid was previously shown to contain sequences homologous to the origin of replication and putative stability loci of pOSU900, a cryptic P. syringae pv. syringae plasmid (Mukhopadhyay et al., 1990; Sundin and Bender, 1994; Sundin et al., 1994). The pattern of restriction fragments generated with BamHI, EcoRI, and SstI were reproduced in separate plasmid preparations of the same strain over a period of months. The banding
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FIG. 1. Graphic representation of plasmid restriction fragments generated by digestion of P. syringae pv. syringae plasmid DNA with EcoRI. Plasmid designations and resistance phenotype follow the strain name in parentheses. The plasmid groups, based on cluster analysis of a similarity matrix generated from restriction fragment data, are shown above each lane. Lanes 1 to 9 contain the following strains: 1, 7B44 (pPSR7-Cur); 2, 8B48 (pPSR11-Cur); 3, FF5.2 (pPSR22-Cur); 4, 3C1 (pPSR10Cur); 5, 2H12 (pPSR9-Cur); 6, 7B12 (pPSR14-Cur Smr); 7, FF3 (pPSR5-Smr); 8, 7B40 (pPSR23-Cur Smr); 9, FF5 (pPSR4-Cur). Linear DNA size standards are indicated at the left.
patterns generated in each plasmid digest suggested that all nine plasmids were related. A diagrammatic representation of the EcoRI banding patterns is shown in Fig. 1. Fragments ranging in size from approximately 1.2 to 18 kb were scored for each plasmid/restriction enzyme combination and the data were converted into a binary matrix for quantitative comparison. Cluster analysis of the data matrix indicated that the plasmids could be assigned to four groups (A–D), with groups A and C containing multiple members which were closely related (Fig. 2a). The restriction fragment data suggested both similarities and distinctions between the plasmids, but provided no information on conserved sequences. Thus, further analyses of the plasmids consisted of Southern hybridization analyses using characterized determinants from P. syringae plasmids. DNA probes specific for the strA-strB Smr genes or the trans-
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posase and resolvase genes of Tn5393 hybridized to the Smr plasmids pPSR5, pPSR14, and pPSR23 (Table 2). Sequences homologous to the ruvZY genes cloned from pPSR1 were present on each of the plasmids (Table 2). Neither IS51 nor IS801 was detected on the plasmids examined in this study (Table 2). Arbitrarily primed PCR analysis. The APPCR technique utilizes oligonucleotide primers in low-stringency PCR reactions to generate patterns of DNA fragments which are strain-specific (Welsh and McClelland, 1990; Williams et al., 1990). Twenty-six different DNA bands, ranging in size from approximately 0.3 to 3.0 kb, were amplified from the nine strains; these bands were numbered (1 to 26) in order of descending size (Fig. 3). The number of DNA bands amplified per strain ranged from two to fifteen (Fig. 3). The data indicated that many bands were detected in more than one strain and that the general pat-
FIG. 2. (a) Dendrogram showing relatedness of nine P. syringae pv. syringae plasmids. The dendrogram was generated from similarity matrices of restriction fragment patterns. Plasmid group designations are shown at the right. (b) Dendrogram of nine P. syringae pv. syringae strains analyzed by the arbitrarily primed polymerase chain reaction technique (AP-PCR). A similarity matrix derived from the DNA banding patterns obtained by APPCR and the IS50 primer was used to generate the dendrogram. Resident plasmids in P. syringae pv. syringae are shown at the right.
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Cur AND Smr PLASMIDS IN P. syringae TABLE 2 DETECTION
OF
SPECIFIC SEQUENCES
IN
P. syringae pv. syringae PLASMIDS Hybridization toa
Plasmid
Groupb
oriV and par
strA-strB
tnpA-tnpR
ruvZY
IS801
IS51
pPSR14 pPSR4 pPSR5 pPSR7 pPSR9 pPSR10 pPSR11 pPSR22 pPSR23
C B C A C A A A D
// // // // // // // // //
// 00 // 00 00 00 00 00 //
// 00 // 00 00 00 00 00 //
// // // // // // // // //
00 00 00 00 00 00 00 00 00
00 00 00 00 00 00 00 00 00
a //, indicates hybridization was observed; 00, indicates no hybridization was observed. The sequences used as probes were: the oriV and par sequences from pOSU900; these were used separately but produced identical results; strA-strB Smr genes and the tnpA and tnpR genes from Tn5393; ruvZY, a determinant from pPSR1 which confers resistance to ultraviolet light; and IS801 and IS51, insertion sequences from P. syringae. b The plasmid groups were assigned based on restriction fragment pattern data (see Fig. 3A).
tern of bands was conserved among groups of strains (Fig. 3). For example, strains 7B44 and 7A7 contain group A plasmids and produced identical banding patterns when their genomic DNA was analyzed by AP-PCR. A data matrix was generated utilizing each strain and scoring for the presence or absence of a particular DNA band. Cluster analysis of the AP-PCR data matrix indicated that all of the P. syringae pv. syringae host strains were clustered into groups which were reflective of the plasmid groups (Fig. 2b).
Interplasmid mobilization of Tn5393. The potential for mobilization of Tn5393 among P. syringae pv. syringae plasmids was assessed using both a compatible and an incompatible plasmid pair. In the incompatible interaction, we monitored for the evolution of a single Cur Smr plasmid following the generation of a strain carrying the two incompatible plasmids, pPSR4 (Cur) and pPSR5 (Smr::Tn5393). The results of two independent matings indicated that both pPSR4 and pPSR5 were initially maintained when the
FIG. 3. Matrix of DNA banding patterns observed for P. syringae pv. syringae strains using the arbitrarily primed polymerase chain reaction technique and the IS50 primer. The DNA bands were numbered from 1 to 26 in order of descending size. ‘‘X’’ indicates the presence of a particular band, and a blank space indicates the absence of that band. The sizes of the amplified DNA fragments are as follows: 1, 3.0 kb; 2, 2.6 kb; 3, 2.5 kb; 4, 2.1 kb; 5, 1.9 kb; 6, 1.5 kb; 7, 1.4 kb; 8, 1.3 kb; 9, 1.25 kb; 10, 1.2 kb; 11, 1.15 kb; 12, 1.1 kb; 13, 1.05 kb; 14, 1.0 kb; 15, 0.95 kb; 16, 0.9 kb; 17, 0.85 kb; 18, 0.8 kb; 19, 0.7 kb; 20, 0.65 kb; 21, 0.55 kb; 22, 0.5 kb; 23, 0.45 kb; 24, 0.4 kb; 25, 0.35 kb; and 26, 0.3 kb.
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FIG. 4. (A) Movement of Tn5393 between two incompatible plasmids in P. syringae pv. syringae FF3. Plasmid designations follow the strain name in parentheses. Lanes 1 to 4 contain the following strains: 1, FF5 (pPSR4); 2, FF3 (pPSR5); 3, FF3 (pPSR4 and pPSR5); 4, FF3 (pPSR4::Tn5393). To simplify presentation of the results, FF5(pPSR4) is shown in lane 1 instead of the actual donor strain 4A39 since the latter contains two cryptic plasmids in addition to pPSR4. Molecular weights (kb) are indicated at the left. (B) Restriction fragment analysis of selected plasmids. Lanes 1 to 3 contain EcoRI digestion products of the following plasmids: 1, pPSR5; 2, pPSR4; 3, pPSR4::Tn5393. Arrow at right indicates EcoRI fragment containing Tn5393. Linear DNA size standards are indicated at the left.
cells were grown with copper and streptomycin selection (Fig. 4A, lane 3). By subculturing the cells in both MGcu sm and MG, we could track the evolution of a single plasmid encoding both copper and streptomycin resistance by correlation with the stability of both determinants in MG broth lacking Cu and Sm. Following a growth period of 32 { 8 generations in MGcu sm broth, Tn5393 was mobilized, presumably by transposition, from pPSR5 to pPSR4; this event was associated with the loss of pPSR5 from the cells (Fig. 4A, lane 4). Analysis of the resulting Cur Smr plasmids by digestion with EcoRI (there are no sites for EcoRI within Tn5393) and hybridization to the strA-strB and tnpA-tnpR probes, indicated that Tn5393 was present on the evolved pPSR4 plasmid in all cases and that it was present in the same 8.5-kb EcoRI restriction fragment (Fig. 4B, lane 3, see arrow). This was correlated with the disappearance of a faintly visible 3.0-kb EcoRI fragment in pPSR4 (Fig. 4B, lane 2, arrow), an event which is consistent with the insertion of the 5.5-kb Tn5393 into this fragment (Sundin and
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Bender, 1995). The 8.5-kb fragment was also cloned into pBluescript (SK/) to generate the recombinant pGWS49; this clone conferred Smr to E. coli and was used to confirm the presence of Tn5393 within the 8.5-kb fragment. In the compatible plasmid interaction, the wild-type P. syringae pv. syringae strain 7C12, which contains two compatible plasmids, pPSR13 (Cur) and pPSR21 (Smr due to Tn5393) was grown for 100 generations in a minimal medium under limited glucose to determine if Tn5393 would move from the large Smr plasmid to the smaller Cur plasmid or to the chromosome. In two separate experiments, there were no observable changes in the size of either plasmid following 100 generations of growth (data not shown). DISCUSSION
In this study, we examined the relatedness of Cur and Smr plasmids sharing a common replicon and their P. syringae pv. syringae hosts. Cluster analysis of the restriction digest patterns indicated that the plasmids could be organized into four groups. Two groups (A and C) contained multiple members which differed only slightly in restriction fragment digest patterns. The conservation of restriction fragments, DNA sequences, and phenotypic traits among the plasmids in these groups suggests that they comprise stable ‘‘cohesive lineages’’ as defined by Riley and Gordon for ColE1 plasmids (Riley and Gordon, 1992). A comparison of the relationships between plasmids and their P. syringae pv. syringae hosts indicated that their phylogenetic trees are quite similar (Figs. 2a and 2b). These results suggest that no recent plasmid transfer events have occurred. The similar phylogenetic trees obtained for the plasmids and their P. syringae hosts and genetic distance between some plasmids (e.g., pPSR7 and pPSR23) and their host strains (7B44 and 7B40, respectively) implies that these plasmids have resided in their hosts for long periods of time. Our previous population study indicated that epiphytic P. syringae pv. syringae strains from ornamental pear could be
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differentiated into two divergent groups (Sundin et al., 1994). Interestingly, specific plasmid profiles were confined to P. syringae pv. syringae host genotypes from a single group (Sundin et al., 1994). Results obtained in the current study confirm these data and imply that the P. syringae pv. syringae plasmids we have studied may be disseminated among groups of related strains but not among more distantly related strains. The compartmentalization of plasmids within specific host genotypes has also been observed in Rhizobium leguminosarum (Young and Wexler, 1988). Epidemiological studies of antibiotic-resistance plasmids have shown that subpopulations of plasmid variants commonly occur within host bacterial populations (e.g., Hopkins et al., 1986; Tran van Hieu et al., 1986; Platt et al., 1988). These variants may contain subtle differences in restriction digest pattern or obvious additions, deletions, or rearrangements of DNA. In many cases, the evolution of multiple antibiotic-resistance plasmids is thought to have occurred via the sequential insertion of transposable elements (LabigneRoussel et al., 1982; Saunders et al., 1986). In this study, the plasmids in individual groups appear to differ only by minor alterations in restriction digest profiles (Fig. 1). In addition, the Cur Smr plasmid pPSR14 may have evolved from the Cur plasmid pPSR9 through the insertion of Tn5393 in a 5.1-kb EcoRI fragment (Fig. 1; lanes 5–6). Thus, the P. syringae pv. syringae plasmids examined in this study may have evolved via mechanisms observed in antibiotic-resistance plasmids inhabiting clinically important bacteria. The plasmid evolution experiments reported here showed that pPSR4 could acquire Tn5393 in vitro. Theoretical and experimental considerations which could promote the evolution of a single multiresistance plasmid from incompatible plasmids have been reported for E. coli (Condit and Levin, 1990). Like Condit and Levin (1990), we did not detect movement of the transposon between compatible plasmids located in the same host cell. The movement of Tn5393 between plasmids is presumably responsible for the presence of the element on plasmids of a variety of sizes within
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natural P. syringae populations (Jones et al., 1991; Sundin et al., 1994). The interplasmid mobilization of Tn5393 mediated by plasmid incompatibility may commonly occur in natural populations; conjugative plasmids containing homologous oriV and par sequences with the plasmid group in this study are common in P. syringae pv. syringae populations (Sundin et al., 1994). Alternatively, Tn5393 could be acquired by a cell from a suicide plasmid, i.e., a Tn5393-containing plasmid which could conjugate to P. syringae but not replicate within the cell. Tn5393 is commonly harbored on large plasmids in plant and soil bacterial populations which co-inhabit nurseries with P. syringae pv. syringae populations (Sundin et al., 1995). Our results indicate that IS51 and IS801, which are associated with plasmid-encoded virulence determinants in other P. syringae pathovars (Soby et al., 1994; Ullrich and Bender, unpublished observations), have not colonized the P. syringae pv. syringae plasmids characterized in the present study. Both IS51 and IS801 have colonized plasmids containing the pOSU900 replicon in other P. syringae pathovars (Murillo and Keen, 1994; Ullrich and Bender, unpublished observations). While plasmids containing the pOSU900 replicon have been detected in at least 16 pathovars (Murillo and Keen, 1994; Sundin et al., 1994), some of the genetic determinants associated with these plasmids are more restricted in distribution. It has been suggested that the conservation of P. syringae plasmids among strains reflects a selective advantage conferred to the P. syringae host (Shaw, 1987). However, many sequences conserved among the P. syringae plasmids have no obvious relationship to pathogenesis (Obukowicz and Shaw, 1985). The UVr determinant conserved among the plasmids in the present study may be important in the survival and propagation of the P. syringae host during the epiphytic phase of its life cycle. Our recent field and growth chamber studies demonstrated that pPSR1, pPSR4, pPSR4::Tn5393a, and pPSR5 did not reduce the epiphytic fitness of P. syringae pv. syringae FF5 in the phylloplane of bean or
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ornamental pear plants (Sundin and Bender, 1994). It is possible that this group and other groups of conserved P. syringae plasmids contain a ‘‘backbone’’ of genes important for ecological fitness in the phylloplane. These plasmids may also become targets into which Cur and Smr genes are inserted. Since not all P. syringae pv. syringae strains contain indigenous plasmids, it would be worthwhile to determine the impact of conserved plasmid-encoded genes on the colonization of particular environmental niches by isogenic plasmidcontaining and plasmid-free strains. ACKNOWLEDGMENTS This research was supported by the Oklahoma Agricultural Experiment Station and National Science Foundation Grant EHR-9108771.
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GARDE, S., AND BENDER, C. L. (1991). DNA probes for detection of copper resistance genes in Xanthomonas campestris pv. vesicatoria. Appl. Environ. Microbiol. 57, 2435–2439. HOITINK, H. A. J., AND SINDEN, S. L. (1970). Partial purification and properties of chlorosis-inducing toxins of Pseudomonas phaseolicola and Pseudomonas glycinea. Phytopathology 60, 1236–1237. HOPKINS, J. D., MAYER, K. H., GILLEECE, E. S., O’BRIEN, T. F., AND SYVANEN, M. (1986). Genetic and physical characterization of IncM plasmid pBWH1 and its variance among natural isolates. J. Bacteriol. 165, 47–52. JONES, A. L., NORELLI, J. L., AND EHRET, G. R. (1991). Detection of streptomycin-resistant Pseudomonas syringae pv. papulans in Michigan apple orchards. Plant Dis. 75, 529–531. KEANE, P. J., KERR, A., AND NEW, P. B. (1970). Crown gall of stone fruit. II. Identification and nomenclature of Agrobacterium isolates. Aust. J. Biol. Sci. 23, 585– 595. KEEN, N. T., TAMAKI, S., KOBAYASHI, D., AND TROLLINGER, D. (1988). Improved broad host-range plasmids for cloning in gram-negative bacteria. Gene 70, 191– 197. KING, E. O., WARD, N. K., AND RANEY, D. E. (1954). Two simple media for the demonstration of pyocyanin and fluorescein. J. Lab. Clin. Med. 44, 301–307. KOBAYASHI, D. Y., TAMAKI, S. J., AND KEEN, N. T. (1990). Molecular characterization of avirulence gene D from Pseudomonas syringae pv. tomato. Mol. Plant– Microbe Interact. 3, 94–102. LABIGNE-ROUSSEL, A., WITCHITZ, J., AND COURVALIN, P. (1982). Modular evolution of disseminated Inc 7-M plasmids encoding gentamicin resistance. Plasmid 8, 215–231. LONG, S. R., AND STASKAWICZ, B. J. (1993). Prokaryotic plant parasites. Cell 73, 921–935. MILLER, J. H. (1972). ‘‘Experiments in Molecular Genetics.’’ Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. MUKHOPADHYAY, P., MUKHOPADHYAY, M., AND MILLS, D. (1990). Construction of a stable shuttle vector for high-frequency transformation in Pseudomonas syringae pv. syringae. J. Bacteriol. 172, 477–480. MURILLO, J., AND KEEN, N. T. (1994). Two native plasmids of Pseudomonas syringae pathovar tomato strain PT23 share a large amount of repeated DNA, including replication sequences. Mol. Microbiol. 12, 941–950. NAGAHAMA, K., YOSHINO, K., MATSUOKA, M., SATO, M., TANASE, S., OGAWA, T., AND FUKUDA, H. (1994). Ethylene production by strains of the plant-pathogenic bacterium Pseudomonas syringae depends upon the presence of indigenous plasmids carrying homologous genes for the ethylene-forming enzyme. Microbiology 140, 2309–2313. NIETO, C., GIRALDO, R., FERNANDEZ-TRESGUERRES, E., AND DIAZ, R. (1992). Genetic and functional analysis of the basic replicon of pPS10, a plasmid specific for Pseudomonas isolated from Pseudomonas syringae pathovar savastanoi. J. Mol. Biol. 223, 415–426.
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Cur AND Smr PLASMIDS IN P. syringae OBUKOWICZ, M., AND SHAW, P. D. (1985). Construction of Tn3-containing plasmids from plant-pathogenic pseudomonads and an examination of their biological properties. Appl. Environ. Microbiol. 49, 468–473. PLATT, D. J., TAGGART, J., AND HERAGHTY, K. A. (1988). Molecular divergence of the serotype-specific plasmid (pSLT) among strains of Salmonella typhimurium of human and veterinary origin and comparison of pSLT with the serotype specific plasmids of S. enteritidis and S. dublin. J. Med. Microbiol. 27, 277–284. RICH, J. J., AND WILLIS, D. K. (1990). A single oligonucleotide probe can be used to rapidly isolate DNA sequences flanking a transposon Tn5 insertion by the polymerase chain reaction. Nucleic Acids Res. 18, 6673–6676. RILEY, M. A., AND GORDON, D. M. (1992). A survey of Col plasmids in natural isolates of Escherichia coli and an investigation into the stability of Col-plasmid lineages. J. Gen. Microbiol. 138, 1345–1352. ROMANTSCHUK, M., RICHTER, G. Y., MUKHOPADHYAY, P., AND MILLS, D. (1991). IS801, an insertion sequence element isolated from Pseudomonas syringae pathovar phaseolicola. Mol. Microbiol. 5, 617–622. SAMBROOK, J., FRITSCH, E. F., AND MANIATIS, T. (1989). ‘‘Molecular Cloning: A Laboratory Manual.’’ Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. SAUNDERS, J. R., HART, C. A., AND SAUNDERS, V. A. (1986). Plasmid-mediated resistance to b-lactam antibiotics in Gram-negative bacteria: The role of in-vivo recyclization reactions in plasmid evolution. J. Antimicrob. Chemother. 18(Suppl. C), 57–66. SHAW, P. D. (1987). Plasmid ecology. In ‘‘Plant Microbe Interactions’’ (T. Kosuge and E. Nester, Eds.), Vol. 2, pp. 3–39. Macmillan Co., New York. SNEATH, P. H. A., AND SOKAL, R. R. (1973). ‘‘Numerical Taxonomy.’’ Freeman, San Francisco. SOBY, S., KIRKPATRICK, B., AND KOSUGE, T. (1993). Characterization of an insertion sequence (IS53) located within IS51 on the iaa-containing plasmid of Pseudomonas syringae pv. savastanoi. Plasmid 29, 135–141. SOBY, S., KIRKPATRICK, B., AND KOSUGE, T. (1994). Characterization of high-frequency deletions in the iaacontaining plasmid, pIAA2, of Pseudomonas syringae pv. savastanoi. Plasmid 31, 21–30. SUNDIN, G. W., AND BENDER, C. L. (1993). Ecological
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and genetic analysis of copper and streptomycin resistance in Pseudomonas syringae pv. syringae. Appl. Environ. Microbiol. 59, 1018–1024. SUNDIN, G. W., AND BENDER, C. L. (1994). Relative fitness in vitro and in planta of Pseudomonas syringae strains containing copper and streptomycin resistance plasmids. Can. J. Microbiol. 40, 279–285. SUNDIN, G. W., AND BENDER, C. L. (1995). Expression of the strA-strB streptomycin resistance genes in Pseudomonas syringae and Xanthomonas campestris and characterization of IS6100 in X. campestris. Appl. Environ. Microbiol. 61, 2891–2897. SUNDIN, G. W., DEMEZAS, D. H., AND BENDER, C. L. (1994). Plasmid and genetic diversity of Pseudomonas syringae isolates differing in exposure to copper and streptomycin bactericides. Appl. Environ. Microbiol. 60, 4421–4431. SUNDIN, G. W., MONKS, D. E., AND BENDER, C. L. (1995). Distribution of the streptomycin-resistance transposon Tn5393 among phylloplane and soil bacteria from managed agricultural habitats. Can. J. Microbiol. 41, 792– 799. TRAN VAN NHIEU, G., GOLDSTEIN, F. W., PINTO, M. E., ACAR, J. F., AND COLLATZ, E. (1986). Transfer of amikacin resistance by closely related plasmids in members of the family Enterobacteriaceae isolated in Chile. Antimicrob. Agents Chemother. 29, 833–837. WELSH, J., AND MCCLELLAND, M. (1990). Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18, 7213–7218. WILLIAMS, J. G. K., KUBELIK, A. R., LIVAK, K. J., RAFALSKI, J. A., AND TINGEY, S. V. (1990). DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18, 6531–6535. YAMADA, T., LEE, P.-D., AND KOSUGE, T. (1986). Insertion sequence elements of Pseudomonas savastanoi: nucleotide sequence and homology with Agrobacterium tumefaciens transfer DNA. Proc. Natl. Acad. Sci. USA 83, 8263–8267. YOUNG, J. P. W., AND WEXLER, M. (1988). Sym plasmid and chromosomal genotypes are correlated in field populations of Rhizobium leguminosarum. J. Gen. Microbiol. 134, 2731–2739. YUCEL, I., BOYD, C., DEBNAM, Q., AND KEEN, N. T. (1994). Two different classes of avrD alleles occur in pathovars of Pseudomonas syringae. Mol. Plant–Microbe Interact. 7, 131–139. Communicated by S. B. Levy
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