Vol. 49, No. 2APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1985, p. 468-4730099-2240/85/020468-06$02.00/0Copyright © 1985, American Society for Microbiology

Construction of Tn3-Containing Plasmids from Plant-PathogenicPseudomonads and an Examination of Their Biological Properties

MARK OBUKOWICZ AND PAUL D. SHAW*

Department of Plant Pathology, University of Illinois, Urbana, Illinois 61801

Received 9 July 1984/Accepted 26 November 1984

Indigenous plasmids isolated from Pseudomonas tabaciATCC 11528(pJPl), Pseudomonas angulata 45(pJP30),and P. tabaci BR2(pBPW1) (M. Obukowicz and P. D. Shaw, J. Bacteriol. 155:438-442, 1983) were labeled withTn3, and the strains were subsequently cured of their respective plasmids. Plasmid-containing and curedisolates caused plant symptoms that were nearly indistinguishable, and the same amount of tabtoxin wasproduced by P. tabaci strains ATCC 11528 and BR2.

The role of plasmids from plant-pathogenic pseudomonadsin the synthesis of various toxins or in eliciting pathogenicsymptoms in host plants has been reviewed (17, 23). Al-though plasmids have been implicated in several diseasescaused by pseudomonads, the only example in which aplasmid has been shown to be involved in inciting diseasesymptoms in host plants is that of oleander gall formationcaused by Pseudomonas savastanoi (3-5). In all other cases,only circumstantial evidence of plasmid involvement intoxin production or pathogenicity has been described (6, 7,9-12, 14, 24).Pseudomonas tabaci, (Pseudomonas syringae pv. tabaci),

causal agent of wildfire disease of tobacco, and Pseu-domonas angulata (P. syringae pv. angulata), causal agentof angular leaf spot of tobacco, are indistinguishable fromeach other except that P. tabaci produces and is resistant totabtoxin, a dipeptide consisting of tabtoxinine-p-lactamlinked to either serine or threonine (27), whereas P. angulatadoes not produce and is sensitive to the toxin (19). Bothorganisms are pathogens on tobacco, but the host for onestrain, P. tabaci BR2, is green beans (26).We have investigated whether tabtoxin synthesis or path-

ogenicity require genes located on cryptic plasmids isolatedfrom P. tabaci ATCC 11528 containing plasmid pJP1 (44.5megadaltons [Mda]) (25), P. tabaci BR2 containing plasmidpBPW1 (30.4 Mda) (23; B. J. Staskawicz, M. Sato, and N. J.Panapoulos, Phytopathology 71:257, 1981), and P. angulata45 containing plasmid pJP30 (49.8 Mda) (25). We considercellular necrosis (pathogenicity) and chlorosis due to tab-toxin to be separate aspects of host symptom expression(28). To determine the role of pJP1 and pBPW1 in tabtoxinsynthesis or pathogenicity or both and the role of pJP30 inpathogenicity, we labeled the three plasmids with the ampi-cillin resistance transposon Tn3 (1). The host strains couldthen be cured of their respective plasmids and then exam-ined for their pathogenicity or their ability to synthesizetabtoxin. Bacterial strains are listed in Table 1 with otherrelevant information.

Bacteria were grown in (i) L broth on 2% L agar (18), (ii)2% Vogel-Bonner (VB) minimal medium (29), or (iii) toxinproduction medium (30) at 26°C. Unless stated otherwise,antibiotics and concentrations used were ampicillin, 300Fig/ml; nalidixic acid, 100 ,ug/ml; rifampin, 100 ,ug/ml (allfrom Sigma Chemical Co.); and streptomycin sulfate, 100Fg/ml (Eli Lilly & Co.).

* Corresponding author.

Conjugation procedures were those described by Chatter-jee and Starr (2). All transconjugants were purified bysingle-colony isolation on second plates containing the ap-propriate antibiotics before plasmid analysis. All controlsshowed a spontaneous reversion frequency of <10'.

Plasmid DNA for restriction endonuclease analysis wasobtained from cesium chloride-ethidium bromide gradients(15). Plasmid DNA for vertical agarose gel analysis wasisolated by using a modified small-scale isolation (30-mlculture) of that same procedure.

Plasmids pJP1, pJP1::Tn3, pJP30, and pJP30::Tn3 weredigested with HindIII (Sigma) and run on horizontal agarosegels (20) to verify the presence of Tn3 in the plasmids.Methods used to select for the transposition of Tn3 onto

pBPW1 and the construction of P. tabaci BR2(pBPW1::Tn3)were described previously (22). Transposition of Tn3 ontopJP1 and pJP30 was performed as follows. PlasmidRSF1010::Tn3 (Strs Sur Ampr; Tn3 inserted in the strepto-mycin phosphotransferase gene) (13) was mobilized by theconjugative plasmid, pBPW1, from E. coli K802(pBPW1;RSF1010::Tn3) into both P. tabaci ATCC 11528(pJP1) (6.1 x10-5 transconjugants per recipient) and P. angulata 45(pJP30)(1.2 x 10-4 transconjugants per recipient) by selecting forAmpr transconjugants. VB rminimal medium was used tocounterselect. Agarose gel electrophoresis of the plasmids(0.7% agarose) and their HindIll digests (0.8% agarose)showed that only RSF1010::Tn3 was acquired by P. tabaciATCC 11528 and P. angulata 45 (data not shown). Portions(0.1 ml) of stationary-phase P. tabaci ATCC 11528(pJP1;RSF1010::Tn3) and P. angulata 45(pJP30; RSF1010::Tn3)cultures were plated on VB minimal medium plus strepto-mycin plus ampicillin to select simultaneously for mainte-nance of Ampr and restoration of Strr. Plates for P. angulata45 contained 200 ,ug of streptomycin per ml, because at 100p.g of streptomycin per ml, spontaneous Strr colonies wereobserved in a control culture without RSF1010::Tn3. In thecase of P. tabaci 11528(pJP1; RSF1010::Tn3), the frequencyof simultaneous appearance of Ampr and Strr and was 6.0 x10-8, whereas for P. angulata 45(pJP30; RSF1010::Tn3) itwas 4.1 x 10-7. No spontaneous Strr isolates were obtainedwith P. tabaci ATCC 11528 or P. angulata 45 isolateslacking RSF1010: :Tn3 (spontaneous reversion frequency,<10-9). Restoration of the Strr phenotype may be due toprecise excision of Tn3 or some other mechanism (deletion).The maintenance of Ampr enabled us to detect the transpo-sition of Tn3 to new sites. Presumably, precise excision ofTn3 from RSF1010::Tn3 and a prior transposition of Tn3

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TABLE 1. Bacterial strains

Strain Plasmid(s) phenotype Chromosomal phenotype Source or reference

E. coli K802 pBPW1 Cryptic Nalr Rif Met- LacY- 22RSF1010::Tn3, Tn3 in Strs Sur Ampr hsdM+ hsdR-

the Strr gene

P. tabaci ATCC 11528 pJP1 Cryptic Rif Spontaneous Rif' mutantof P. tabaci ATCC11528

pJP1 Cryptic Rif This laboratoryRSF1010::Tn3 Strs Sur AmprpJP1::Tn3, Tn3 in the Ampr Rif This laboratory

fourth- or sixth-largest HindlIl frag-ment

Rifr This laboratory

P. tabaci BR2 pBPW1 Cryptic Rif Spontaneous Rif' mutantof strain obtainedfrom N. Panopoulos

Rifr This laboratorypBPW1::Tn3 Ampr Rift 22

P. angulata 45 pJP30 Cryptic Rift This laboratorypJP30 Cryptic Rift This laboratoryRSF1010::Tn3 Strs Sur AmprpJP30::Tn3 Ampr Rift This laboratoryRSF1010 Strr SurRSF1010 Strr Sur Rif This laboratory

1 2 3 4 5 6 7

14.1

6.1

4.1

1.61.4

FIG. 1. Agarose gel (0.8%) showing HindlIl restriction endo-nuclease digests of two P. tabaci ATCC 11528(pJP1::Tn3) isolatesand one P. angulata 45(pJP30::Tn3; RSF1010) isolate. pJP1 is cuteight times by HindlIl, giving fragments of 10.8, 9.4, 8.0, 7.1, 2.8,2.3, 1.5, and 1.4 Mda (25). pJP30 is cut 16 times by Hindlll, givingfragments of 10.8, 9.0, 7.1, 6.7, 2.4, 2.4, 2.1, 2.1, 2.0, 1.9, 1.8, 1.5,1.0, 0.9, 0.7, and 0.7 Mda (25). Lane 1, A DNA digested withHindlIl; lane 2, P. tabaci ATCC 11528 with Tn3 transposed into thefourth-largest HindlIl fragment of pJP1; lane 3, P. tabaci ATCC11528 with Tn3 transposed into the sixth-largest HindIll fragment ofpJP1; lane 4, P. tabaci ATCC 11528(pJP1) control; lane 5, P.

into pJP1 and pJP30 occurred. VB minimal medium wasused to eliminate those transposition events into the chro-mosome that would cause auxotrophic mutants. SinceRSF1010 is a high-copy-number plasmid (8, 21), it is possiblethat restoration of Strr occurred in only one plasmid copy.Therefore, two additional single-colony isolations were madeon VB agar plus streptomycin to exclude cells that containedparental RSF1010::Tn3. Of the 144 randomly chosen AmprStrr P. tabaci ATCC 11528 colonies, 13 retained Ampr.Similarly, of the 204 randomly chosen Ampr Strr P. anglilata45 colonies, 28 retained Ampr. Thus, 9.0% of the P. tabaciATCC 11528 colonies and 13.7% of the P. angulata 45colonies that were selected for maintenance of Ampr andrestoration of Strr presumably contained Tn3 in anothergenomic location. HindIII digests carried out on plasmidsfrom two P. tabaci ATCC 11528 isolates showed that Tn3had transposed into pJP1 at two different sites (Fig. 1). Also,a HindlIl digest of the plasmid from a single P. angulata 45isolate showed that Tn3 transposed into pJP30.For plasmid curing, cells (3 x 103) from stationary-phase

L broth cultures were transferred to 30 ml of fresh L brothcontaining a range of concentrations of various curing agents(final L broth pH = 7.6). Individual colonies (200) that grewup after 48 h were then transferred onto L agar plus

angulata 45 with Tn3 transposed into the second-largest HindIllfragment of pJP30; lane 6, P. angulata 45(pJP30; RSF1010) control;lane 7, P. angulata 45(pJP30) control (RSF1010 is not present). Thesmearing in lanes 5 and 6 is due to uncut RSF1010, which does notrun as a discrete band (C. Muster, personal communication). Notethe disappearance of a single band in lanes 2, 3, and 5 and thereappearance of a new band ca. 3 Mda larger in size.

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FIG. 2. DNA-DNA hybridization of 32P-labeled plasmid probeswith HindIll-digested chromosomal DNA from cured strains of P.tabaci ATCC 11528, P. tabaci BR2, and P. angulata 45(RSF1010).(A) Agarose gel before blotting and hybridization. Lane 1, cured P.tabaci ATCC 11528 DNA; lane 2, cured P. tabaci ATCC 11528DNA plus one copy equivalent of pJP1::Tn3; lane 3, controlpJP1::Tn3 plasmid DNA digested with HindIll; lane 4, cured P.tabaci BR2 DNA; lane 5, cured P. tabaci BR2 DNA plus one copyequivalent of pBPW1::Tn3; lane 6, control pBPW1::Tn3 plasmidDNA digested with Hindlll; lane 7, cured P. angulata 45(RSF1010)DNA (RSF1010 is still present in the cured strain); lane 8, cured P.angulata 45(RSF1010) DNA plus one copy equivalent of pJP30: :Tn3(RSF1010 is present along with pJP30::Tn3 in the plasmid DNA);lane 9, control pJP30: :Tn3 and RSF1010 plasmid DNA digested withHindlIl. Covalently closed circular RSF1010 is present as a smearbetween the fourth- and fifth-largest HindIII bands of pJP30::Tn3.Open circular RSF1010 comigrates as a less diffuse smear with thethird-largest HindIll fragment of pJP30::Tn3 (cf. Fig. 1, lanes 5through 7). (B) Results of hybridization. Self-hybridization occurredwith each of the plasmid probes (lanes 3, 6, and 9). The smear withpJP30::Tn3; RSF1010 in lane 9 is due to RSF1010 in the probehybridizing to covalently closed circular and open circular smeared

ampicillin, L agar plus streptomycin, and L agar withoutantibiotics. The number of Amps (loss of Tn3-containingplasmids) or Strs (loss of RSF1010) colonies was determined24 h later. P. tabaci ATCC 11528 was cured of pJP1::Tn3and RSF1010 simultaneously with ethidium bromide (10jig/ml) at a frequency of 0.5% (1/200). P. angulata was curedof pJP30::Tn3 with acridine orange (100 ,ug/ml) at a fre-quency of 1.2% (2/163). P. tabaci BR2 was cured ofpBPW1: :Tn3 with 2% sodium dodecyl sulfate at a frequencyof 1.0% (2/200). The ability of the compounds to causeplasmid loss was thus somewhat strain specific or plasmidspecific or both. In each instance, the colony morphology ofparental and cured strains was indistinguishable.The removal of plasmid DNA from the putative cured

strains was verified by DNA-DNA hybridizations betweentotal and plasmid probe DNA. Total DNAs from curedstrains ofP. tabaci ATCC 11528 and BR2 and P. angulata 45were obtained from uncleared lysates precipitated directlywith polyethylene glycol and purified on cesium chloride-ethidium bromide gradients (15). pJP1: :Tn3 and pBPW1::Tn3plasmid DNA probes were isolated from P. tabaci ATCC11528 and BR2, respectively, that had been cured ofRSF1010. In the case of P. angulata 45, RSF1010 waspresent in addition to pJP30::Tn3 because of the failure toselectively cure strain 45 (pJP30::Tn3; RSF1010) ofRSF1010.DNA from cured strains of P. tabaci ATCC 11528 (4.2 ,ug),P. tabaci BR2 (5.0 ,ug), and P. angulata 45(RSF1010) (3.1,ug) was digested with HindIll and subjected to horizontalagarose gel electrophoresis. One copy equivalent of thecorresponding plasmid DNA was added to a second DNAsample, digested with HindlIl, and subjected to electropho-resis in a second slot. For controls, corresponding plasmidprobe DNA (2 ,ug) was digested with HindlIl and run in athird slot. Plasmids pJP1::Tn3, pBPW1::Tn3, and apJP30::Tn3-RSF1010 mixture were labeled by nick translation with[a-32P]dATP (Amersham Corp.) and hybridized to the ap-propriate DNAs on nitrocellulose sheets (25). Filters wereexposed for 24 h to Kodak No-Screen X-ray film. DNA-DNA hybridization data showed no detectable hybridizationof pJP1::Tn3, pBPW1::Tn3, or pJP30::Tn3 plasmid probes,respectively, with total DNA from cured strains of P. tabaciATCC 11528 and BR2 and P. angulata 45(RSF1010) (Fig. 2Aand B). One copy equivalent of plasmid DNA added tochromosomal DNA from the cured strains could be detected(Fig. 2A and B).

Plasmid-containing and cured strains of P. tabaci ATCC1i528 and BR2 were assayed for tabtoxin production in

RSF1010. In all cases, one copy equivalent of plasmid DNA addedto chromosomal DNA from the cured strains was detected (lanes 2,5, and 8). Only the larger plasmid HindIII bands were detected. Thefirst, fourth, and fifth bands of pJP1::Tn3 are evident (lane 2). Thefirst, second, and third bands of pBPW1::Tn3 are evident; the fifthand sixth bands are very faint (lane 8). As with lane 9, the smearedhybridization in the middle of lane 8 is due to RSF1010 in the probehybridizing to covalently closed circular and open circular smearedRSF1010 added along with pJP30::Tn3 to the DNA. In no case wasany hybridization with plasmid Hindlll fragments detected in chro-mosomal DNA Hindlll fragments from cured P. tabaci ATCC 11528or BR2 or P. angulata 45(RSF1010) (lanes 2, 4, and 7). In the caseof cured P. angulata 45(RSF1010), hybridization occurred, as ex-pected, with open circular RSF1010 that banded with chromosomalDNA in cesium chloride-ethidium bromide gradients (lane 7). Thebackground smears were due to the long exposure time used (24 h)to enhance any detection of plasmid DNA in total DNA from thecured strains.

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FIG. 3. Symptom expression of tobacco Samson NN leaves injected with plasmid-containing and cured strains of P. tabaci ATCC 11528and BR2. (A through C) Leaves were injected with P. tabaci ATCC 11528(pJP1) (leaves 1) or cured P. tabaci ATCC 11528 (leaves 2). Cultureswere injected undiluted (A) or at dilutions of 10-2 (B) or i0-' (C). (D through F) Leaves were injected with P. tabaci BR2(pBPW1) (leaves1), cured P. tabaci BR2 (leaves 2), P. tabaci BR2(pBPW1; RSF1010) (leaves 3), or P. tabaci BR2 containing pBPW1 and RSF1010 mobilizedback into the cured strain (leaves 4). Cultures were injected undiluted (D) or at dilutions of 10-1 (E) or 10-2 (F).

liquid toxin production medium by using the Salmonellaoverlay technique (25). Assays were performed in triplicateat 24, 48, 72, and 96 h. Supernatant solutions from culturesof both plasmid-containing and cured strains of P. tabaciATCC 11528 and BR2 gave nearly identical diameters ofSalmonella growth inhibition (data not shown).For pathogenicity assays, plants were grown in a growth

chamber (day, 14 h, 28°C; night, 10 h, 20°C; 26.9 x 103 lx).Strains containing their native plasmids and derivativescured of their respective plasmids were assayed for patho-genicity in both tobacco and green bean leaf tissue. Bacterialsuspensions (ca. 100 [L of 2.0 x 107 to 1.4 x 108 cells per ml;A660 = 0.1) in sterile distilled water made from overnightcultures grown on VB agar were injected into six intercostalareas of the first fully expanded leaves of tobacco (Nicotianatabacum L. Samson NN and N. tabacum L. Havana 38) andthe first fully expanded trifoliate leaves of green beans(Phaseolus vulgaris L. Top Crop) (16). All injections were

performed in duplicate on two different plants. Plasmid-con-taining and cured strains were injected into adjacent leavesof the same plant. Symptom expressions were examined 1week after bacterial injections.

Pathogenicity was assayed in both host and nonhostplants. Symptoms of leaf necrosis began to appear within 24to 48 h on both host and nonhost plants injected withsufficient bacteria. After 1 week, pJP1-containing and curedstrains of P. tabaci ATCC 11528 gave identical symptoms atevery dilution on Samson NN tobacco and green bean leaves

(Fig. 3A through C and Fig. 4A through C). Cured P. tabaciBR2 was slightly more virulent on tobacco than P. tabaciBR2 containing pBPW1 (Fig. 3D through F). Additionalevidence that loss of pBPW1 is associated with slightlyincreased virulence comes from conjugation studies in whichpBPW1 and RSF1010 were mobilized back into cured BR2.When pBPW1 was reinserted back into cured BR2, thepathogenicity characteristics of the strain reverted back tothat of the parent containing pBPW1 (Fig. 3D through F). Toshow that the presence of RSF1010 did not influence patho-genicity, P. tabaci BR2(pBPW1; RSF1010) (strain trans-formed with RSF1010) was also injected into Samson NNtobacco plants. At all dilutions, P. tabaci strainsBR2(pBPW1), BR2(pBPW1; RSF1010), and BR2 withpBPW1 and RSF1010 reinserted into the cured strain showednearly identical symptoms when injected into tobacco leaves.These injections were repeated three additional times toverify the difference in virulence between cured and pBPW1-containing strains. Two cured isolates obtained independ-ently were also used. In each case, the cured strains were

slightly more virulent than the pBPW1-containing strains. Incontrast, green bean leaves injected with cured P. tabaciBR2 or P. tabaci BR2(pBPW1) showed nearly identicalsymptoms at every dilution (Fig. 4D through G).As with P. tabaci ATCC 11528, P. angulata strains

45(RSF1010) (cured of pJP30), 45(pJP30; RSF1010), and45(pJP30) gave nearly identical symptoms when injected intoeither Samson NN tobacco or green bean leaves (data not

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FIG. 4. Symptom expression of green bean leaves injected with plasmid-containing and cured strains of P. tabaci ATCC 11528 and BR2.(A through C) Leaf injections with P. tabaci ATCC 11528(pJP1) (leaves 1) and cured P. tabaci ATCC 11528 (leaves 2). Cultures were injectedundiluted (A) or at dilutions of 10-1 (B) or 10-2 (C). Symptom expression is nearly absent. (D through G) Leaves were injected with P. tabaciBR2(pBPW1) (leaves 1) or cured P. tabaci BR2 (leaves 2). Cultures were injected undiluted (D) or at dilutions of 10-2 (E), 10-3 (F), or 10-4(G).

shown). The presence of RSF1010 did not influence viru-lence.The pathogenicity results obtained with the somewhat

resistant tobacco variety Samson NN were verified by assayof the more susceptible variety (R. Durbin, personal com-munication), Havana 38. Results were similar to thoseobtained with Samson NN; however, Havana 38 was 10times more sensitive to P. tabaci ATCC 11528 and P.angulata 45. Cured P. tabaci BR2 was again somewhat morevirulent than pBPW1-containing P. tabaci BR2. Havana 38was 103 times more sensitive to P. tabaci BR2.

Results of experiments with the cured pseudomonadstrains that we have constructed lead to two conclusions.pJP30, pJP1, and pBPW1 do not encode genes essential forpathogenicity (Fig. 3 and 4), and pJP1 and pBPW1 do notencode genes essential for tabtoxin synthesis. This workdoes not prove that plasmids are never involved in patho-genesis in these diseases. Rather, these three plasmids donot encode pathogenic determinants in the strains examined.

We thank S. Falkow, J. Gardner, N. Panopoulos, and S. Ries forproviding bacterial cultures and J. Johnston, C. Muster, and J.Gardner for helpful discussions during the course of the research.

LITERATURE CITED

1. Campbell, A., D. E. Berg, D. Botstein, E. M. Lederberg, R. P.Novick, P. Starlinger, and W. Szybalski. 1979. Nomenclature oftransposable elements in prokaryotes. Gene 5:197-206.

2. Chatterjee, A. K., and M. P. Starr. 1973. Gene transmissionamong strains of Erwinia amylovora. J. Bacteriol. 116:1100-1106.

3. Comai, L., and T. Kosuge. 1980. Involvement of plasmiddeoxyribonucleic acid in indoleacetic acid synthesis in Pseu-domonas savastonoi. J. Bacteriol. 143:950-957.

4. Comai, L., and T. Kosuge. 1982. Cloning and characterization of

iaaM, a virulence determinant of Pseudomonas savastanoi. J.Bacteriol. 149:40-46.

5. Comai, L., G. Surico, and T. Kosuge. 1982. Relation of plasmidDNA to indoleacetic acid production in different strains ofPseudomonas syringae pv. savastanoi. J. Gen. Microbiol.128:2157-2163.

6. Curiale, M. S., and D. Mills. 1977. Detection and characteriza-tion of plasmids in Pseudomonas glycinea. J. Bacteriol.131:224-228.

7. Currier, T. C., and M. K. Morgan. 1983. Plasmids of Pseu-domonas syringae: no evidence of a role in toxin production orpathogenicity. Can. J. Microbiol. 29:84-89.

8. de Graaff, J., J. H. Crosa, F. Heifron, and S. Falkow. 1978.Replication of the nonconjugative plasmid RSF1010 in Escheri-chia coli K-12. J. Bacteriol. 134:1117-1122.

9. Gantotti, B. V., S. S. Patil, and M. Mandel. 1979. Apparentinvolvement of a plasmid in phaseotoxin production by Pseu-domonas phaseolicola. Appl. Environ. Microbiol. 37:511-516.

9a.Gonzalez, C. F., and R. H. Olsen. 1981. Conjugal transfer of anindigenous plasmid of Pseudomonas syringae. Phytopathology71:220.

10. Gonzalez, C. F., and A. K. Vidaver. 1979. Syringomycin pro-duction and holcus spot disease of maize: plasmid-associatedproperties in Pseudomonas syringae. Curr. Microbiol. 2:75-80.

11. Gonzalez, C. F., and A. K. Vidaver. 1980. Restriction enzymeanalysis of plasmids from syringomycin-producing strains ofPseudomonas syringae. Phytopathology 70:223-225.

12. Gross, D. C., and A. K. Vidaver. 1981. Transformation ofPseudomonas syringae with nonconjugative R plasmids. Can. J.Microbiol. 27:759-765.

13. Heffron, F., C. Rubens, and S. Falkow. 1975. Translocation of aplasmid DNA sequence which mediates ampicillin resistance:molecular nature and specificity of insertion. Proc. Natl. Acad.Sci. U.S.A. 782:3623-3627.

14. Jamieson, A. F., R. L. Bieleski, and R. E. Mitchell. 1981.Plasmids and phaseolotoxin production in Pseudomonas syrin-gae pv. phaseolicola. J. Gen. Microbiol. 122:161-165.

15. Johnston, J. B., and I. C. Gunsalus. 1977. Isolation of metabolic

472 NOTES

on January 30, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

VOL. 49, 1985 NOTES 473

plasmid DNA from Pseudomonas putida. Biochem. Biophys.Res. Commun. 75:13-17.

16. Klement, Z. 1963. Method for the rapid detection of the patho-genicity of phytopathogenic pseudomonads. Nature (London)199:299-300.

17. Lacy, G. H., and J. V. Leary. 1979. Genetic systems inphytopathogenic bacteria. Annu. Rev. Phytopathol. 17:181-202.

18. Lennox, E. S. 1955. Transduction of linked genetic characters ofthe host bacteriophage P1. Virology 1:190-206.

19. Lucas, G. B. 1975. Diseases of tobacco, 3rd ed. BiologicalConsulting Association, Raleigh, N.C.

20. Meyers, J. A., D. Sanchez, L. P. Elwell, and S. Falkow. 1976.Simple agarose gel electrophoretic method for the identificationand characterization of plasmid deoxyribonucleic acid. J. Bac-teriol. 127:1529-1537.

21. Nagahari, K., and K. Sakaguchi. 1978. RSF1010 plasmid as apotentially useful vector in Pseudomonas species. J. Bacteriol.133:1527-1529.

22. Obukowicz, M., and P. D. Shaw. 1983. Tn3 labeling of a crypticplasmid found in the plant pathogenic bacterium Pseudomonastabaci and mobilization of RSF1010 by donation. J. Bacteriol.155:438 442.

23. Panopoulos, N. J., and B. J. Staskawicz. 1981. Genetics ofproduction, p. 79-107. In R. D. Durbin (ed.), Toxins in plant

disease. Academic Press, Inc., New York.24. Panopoulos, N. J., B. J. Staskawicz, and D. Sandlin. 1979.

Search for plasmid-associated traits and for a cloning vector inPseudomonas phaseolicola, p. 365-371. In K. N. Timmis andA. Puhler (ed.), Plasmids of medical, environmental, and com-mercial importance. Elsevier/North-Holland Publishing Co.,Amsterdam.

25. Piwowarski, J. M., and P. D. Shaw. 1982. Characterization ofplasmids from plant pathogenic pseudomonads. Plasmid 7:85-94.

26. Ribeiro, R. de L. D., D. J. Hagedorn, R. D. Durbin, and T. F.Uchytil. 1979. Characterization of the bacterium inciting beanwildfire in Brazil. Phytopathology 69:208-212.

27. Stewart, W. W. 1971. Isolation and proof of structure of wildfiretoxin. Nature (London) 229:174-178.

28. Turner, J. G. 1981. Tabtoxin, produced by Pseudomonastabaci, decreases Nicotiana tabacum glutamine synthetase invivo and causes accumulation of ammonia. Physiol. Plant Pa-thol. 19:57-67.

29. Vogel, H., and D. M. Bonner. 1956. Acetylornithinase of Esch-erichia coli: partial purification of some properties. J. Biol.Chem. 218:97-106.

30. Wooley, D. W., R. B. Pringle, and A. Braun. 1952. Isolation ofthe phytopathogenic toxin of Pseudomonas tabaci, an antago-nist of methionine. J. Biol. Chem. 197:409-415.

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