APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1986, p. 323-3270099-2240/86/020323-05$02.00/0Copyright © 1986, American Society for Microbiology

Vol. 51, No. 2

Generation and Characterization of Tn5 Insertion Mutations inPseudomonas syringae pv. tomato

DIANE A. CUPPELSAgriculture Canada, Research Centre, London, Ontario, Canada N6A 5B7

Received 3 September 1985/Accepted 28 October 1985

TnS-induced insertion mutations were generated in the Pseudomonas syringae pv. tomato genome by matingthis plant pathogen with an Escherichia coli strain carrying the suicide plasmid vector for TnS, pGS9. Kmrtransconjugants occurred at frequencies ranging from 2 x 10-7 to 9 X 10-6; approximately 5.5% of thesetransconjugants were also Cmr, indicating the presence of additional pGS9 DNA sequences. Approximately 1%

of the Kmr Cms mutants were auxotrophic. Southern blot analysis revealed that the TnS element had insertedinto one unique site on the chromosome for each Kmr Cms transconjugant examined. Physical and genetic testsof TnS-induced auxotrophs showed that TnS mutations in P. syringae pv. tomato were very stable and thatsecondary transposition of TnS or its insertion sequence ISSO was a rare event. Nine of 920 Kmr Cmstransconjugants screened on tomato seedlings either were avirulent or produced very mild symptoms. Each ofthe virulence mutants was the result of a unique single-site Tn5 insertion. Five mutants also failed to induce a

hypersensitivity reaction on tobacco.

Pseudomonas syringae pv. tomato (Okabe 1933) Young etal. 1978 causes bacterial speck, a serious disease problem onfresh market and processing tomatoes. On leaves this dis-ease is characterized by necrotic lesions surrounded bychlorotic halos. To date, the only known host for P. syringaepv. tomato is the tomato plant. Little is known about thegenetics of this pathogen; even less is understood about themolecular mechanisms by which it induces plant disease.Recent advances in recombinant DNA technology have

accelerated genetic analyses of some of the other plantpathogenic bacteria, including other leaf spotters (1, 6, 9, 17,24). An important, versatile tool in this new approach is thetransposon (transposable genetic element) Tn5 (13). Itstransposition into bacterial DNA is random and results insingle-site nonleaky polar mutations with a selectable pheno-type (kanamycin and neomycin resistance). TnS mutagenesisgreatly facilitates the cloning of genes or regions of thechromosome for which direct selection is not possible. It canalso accelerate the construction of correlated physical andgenetic maps of the cloned DNA segments (11).

Several vectors have been constructed for use in thegeneration of TnS insertion mutations (11). The most com-monly used vectors have been pJB4J1 and its derivatives,which are cointegrates of an IncP-type plasmid andbacteriophage Mu DNA. The recently described suicideplasmid vector pGS9, consisting of a pl5A replicon andN-type conjugal transfer genes, has a definite advantage overpJB4J1 in that it does not contain Mu DNA and thus is notlikely to induce secondary genetic changes in the TnS-mutagenized genome (20). It has been successfully used inthe mutagenesis of Rhizobium meliloti, R. leguminosarum,and R. japonicum (19, 20).

In the present study TnS was introduced through vectorpGS9 into the P. syringae pv. tomato genome. The efficiencyof Tn5 delivery and vector loss are similar to those reportedfor the Rhizobium spp. (19, 20). TnS-induced insertionmutations occurred randomly and were very stable in P.syringae pv. tomato. Secondary transposition was not ob-served. Auxotrophic and virulence mutants, both present ata frequency of approximately 1%, were isolated and charac-terized.

(A preliminary account of this work was presented at the6th International Conference on Plant Pathogenic Bacteria,Beltsville, Md., June 1985).

MATERIALS AND METHODS

Bacterial strains and plasmids. The bacterial strains andplasmids used in this study are listed in Table 1. PlasmidspACYC184 and pCU1 were carried in Escherichia coliHB101; plasmid pGS9 was carried in E. coli WA803.Media and growth conditions. P. syringae pv. tomato

strains were grown on nutrient broth-yeast extract medium(NBY) (25) at 25°C. E. coli strains were grown at 37°C on LBmedium (15) containing the appropriate antibiotics. Tn5-induced mutants of P. syringae pv. tomato were stored at-73°C in NBY broth containing 15% glycerol.Vogel-Bonner minimal medium (10), solidified with 1.5%

purified agar (Difco Laboratories), was used to screen forauxotrophy among the transconjugants. The specific nutri-tional requirements of the auxotrophs were determined bythe procedure described by Davis et al. (10).When appropriate, the media were supplemented with one

or more of the following antibiotics (in micrograms permilliliter): kanamycin, 50; streptomycin, 50; rifampin, 50;chloramphenicol, 100; carbenicillin, 100; and tetracycline,25.

Bacterial matings. Both the P. syringae pv. tomato recip-ient strains and the E. coli donor strain were grown in LBbroth at 29°C to a density of about 5 x 108 CFU/ml.Approximately 5 x 108 CFU of donor were mixed with 2.5 x109 CFU of recipient and collected by filtration on a HAWPmembrane filter (pore size, 0.45 ,um; Millipore Corp.). Thefilter was incubated on LB agar at 29°C for 16 to 18 h beforethe bacteria were washed off into 10 mM potassium phos-phate buffer (pH 7.2). Serial dilutions of this bacterialsuspension were spread over the surfaces of the appropriateselective media, and the plates were incubated at 25°C for 96h. The same procedure was followed for the donor andrecipient controls.

Isolation of DNA. Whole-cell DNA was isolated from P.syringae pv. tomato strains by the method of Shepard andPolisky (23). Plasmids pGS9, pCU1, and pACYC184 were

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TABLE 1. Bacterial strains and plasmids

PRelevant Source orcharacteristics reference

StrainP. syringae pv. tomatoDC 21, 22, 24, 39, 51 Wild type This labo-

52, 53, 54, 73, 76 ratorySM78-3 Wild type S. McCarter1071 Wild type J. LindemannCNBP1427a Wild type R. SamsonPDDCC4358b Wild type D. DyeNCPPB880C Wild type D. DyeDC3000 Spontaneous Rif' This labo-

derivative of DC52 ratory

E. coliWA803 met thi 26HB101 pro leu thy thi 2

PlasmidspGS9 Cmr Kmr, pl5A 20

replicon, N-tra,dTnS donor

pCU1 Smr Cbr, N group 20replicon, N-trad

pACYC184 Cmr TCr, pl5A 4replicon

a La Collection Nationale de Bacteries PhytopathogEnes (France).b Plant Disease Division Culture Collection (New Zealand).c National Collection of Plant Pathogenic Bacteria (England).d N-tra, Functional conjugal transfer system.

purified from the appropriate E. coli strains by a combinationof the alkaline lysate procedure of Portnoy and White (7) andethidium bromide-cesium chloride gradient centrifugatiop(15). Purified DNA was stored in TE buffer (10 mM Tris, 0.1mM EDTA, pH 8.0) at 4°C.

Detection of plasmid DNA. Single colonies of P. syringaepv. tomato were screened for the presence of plasmid DNAby the rapid detection procedure of Kado and Liu (12). Thealkaline lysate was incubated at 60°C for 60 min. PlasmidDNA was subjected to electrophoresis in a 0.45% agarosegel with Tris-borate buffer (2.2 V/cm for 18 h) (10). The gelwas stained with eithidium bromide (0.5 ,ug/ml), visualizedwith a 254-nm UV transilluminator (C61; UV Products), andphotographed with a Polaroid MP-4 Land camera.

Restriction endonuclease digestion and gel electrophoresis.Restriction endonucleases EcoRI, PvuI,and PstI were pur-chased from Bethesda Research Laboratories, Inc., orBoehringer Mannheim Biochemicals. Whole-cell DNA wasdigested under the conditions suggested by the manufacturerof the restriction enzyme and subjected to electrophoresis ina 0.8% agarose gel with Tris-borate buffer (2 V/cm for 18 h).DNA ifiter hybridization. After electrophoresis, restriction

endonuclease-digested DNA was transferred to nitrocellu-lose paper by the method of Southern (15). Purified plasmidDNAs labeled with 32P by nick translation (18) were hybrid-ized to the nitrocellulose blots by the procedure of Cohen etal. (5). Autoradiograms were prepared by exposing KodakXAR-5 X-ray film (in the presence of Du Pont CronexLightning-Plus intensifying screens) to the blots for variouslengths of time at -700C (15).

Plant pathogenicity and hypersensitivity tests. Bacteriagrown overnight on NBY agar grid plates at 25°C weresuspended in 10 mM potassium phosphate buffer (pH 7.2)containing 1 mM magnesium sulfate to a cell density ofapproximately 107 CFU/ml. Fourteen-day-old tomato seed-

lings (Lycopersicon esculentum Mill. 'Bonny Best') wereinoculated by gently rubbing the first true leaves with asterile cotton swab moistened with the bacterial suspension.Inoculated seedlings were incubated for 7 days in a growthchamber under a 15-h light (1,400 microeinsteins/m2 per s)(24C)-9-h dark (20°C) cycle. Relative humidity was set at80%. Pathogenic strains produced necrotic lesions sur-rounded by chlorotic halos within 4 to 5 days of inoculation.The hypersensitivity reaction on tobacco leaves

(Nicotiana tabacum L. 'White Gold') was tested as previ-ously described (8).

RESULTSTransfer of pGS9 to P. syringae pv. tomato. In matings with

E. coli WA803(pGS9), P. syringae pv. tomato DC3000acquired kanamycin resistance (Kmr) at frequencies rangingfrom 2 x 10-7 to 9 x 10-6 per recipient cell. Three (DC54,DC39, and CNPB1427) of 14 other P. syringae pv. tomatostrains tested also obtained TnS in crosses with WA803(pGS9) but at lower frequencies (1.7 x 10-7 to 1.4 x 10-8).In comnparison, spontaneous mutation by P. syringae pv.tomato to kanamycin resistahce on LB plates containing 50,g of kanamycin per ml occurred at a frequency of 101-.TnS also codes for resistance to streptomycin, a phenotypeexpressed in Rhizobium spp. but not in E. coli (21). Every P.syringae pv. tomato Kmr transconjugant tested was resistantto this antibiotic. Replica plating onto Vogel-Bonner mini-mal-Km-Cm agar of 974 DC3000 transconjugants revealedthat 5.5% of the strains were Cmr and thus had inheritedadditional pGS9 DNA sequences. Five randomly chosenKmr Cmr transconjugants and 10 Kmr Cms transconjugantswere screened for the presence of pGS9 (30.5 kilobases).The only plasmid found in all 15 was the indigenous DC3000plasmid (ca. 68 kilobases).

Physical analysis of P. syringae pv. tomato Tn5 transposi-tions. The distribution and number of TnS elpments in theDNA of P. syringae pv. tomato Kmr Cms transconjugantswas detertmined by Southern blot analysis. PvuI-digestedtotal cellular DNA from seven transconjugants was hybrid-

1 2

93 4 5 6 7 8 9 Kb

-14.30 I-* 12.7

40 4D 4 11.9_-. 9.5

FIG. 1. Southern blot analysis of PvuI-digested total cellularDNA from TnS-induced mutants of P. syringae pv. tomato. Blots ofdigested DNA were hybridized with 32P-labeled pGS9, the TnSvector. Lane 1, pGS9 (undigested); lane 2, strain DC3000 (wildtype); lanes 3 to 9, Kmr Cms transconjugants DC3281, DC3381,DC3132, DC3244, DC3238, DC3195, and DC3121. The numbers onthe right refer to size markers (in kilobases [kb]) prepared bydigesting lambda DNA with PvuI.

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ized with 32P-labeled pGS9 DNA (Fig. 1). Since Tn5 does notcontain a recognition site for PvuI (11), only one fragmentfrom each transconjugant DNA digest should hybridize tothe probe. Furthermore, since Tn5 has a low insertionalspecificity, the Tn.5-containing probe is likely to hybridize todifferent PvuI fragments for each transconjugant examined.Although all seven strains contained a single Tn5 element,there were two instances where the hybridizing PvuI frag-ments for two transconjugant strains appeared to be thesame size: strains 3238 and 3121 and strains 3195 and 3244.Whether or not the Tn5 insertion sites for these strains were

the same was determined by digesting the whole-cell DNAsfrom these strains with either EcoRI or PstI (Fig. 2). EcoRI(11), like PvuI, did not cleave Tn5 and thus the Tn5 probehybridized with just one fragment for each strain. PstIcleaved Tn5 four times (11), resulting in three internalfragments of set size and two boundary fragments of varyingsizes. EcoRI digestion clearly distinguished strains 3195 and3244 but not strains 3238 and 3121 (Fig. 2A); PstI digestionseparated strains 3238 and 3121 (Fig. 2B). Thus, the TnSinsertion site for all seven transconjugants was unique.Additional evidence that no part of pGS9, other than the Tn5element, had inserted into the genome of these seven Kmr

A B1 2 3 4 1 2 3

* .p.30

_b

Kb

-.21.2

- 7.4

= 5.85.64.9

- 3.5

FIG. 2. Southern blot analysis of (A) EcoRl- or (B) Pstl-digestedtotal cellular DNA from TnS-induced mutants of P. syringae pv.

tomato. Blots of digested DNA were hybridized with 32P_labeled

pGS9. Lane Al, Strain DC3244; lane A2, strain DC3195; lanes A3

and Bi, strain DC3121; lanes A4 and B2, strain DC3238; lane B3,

pGS9. The numbers on the ri'ght refer to size markers (in kilobases

[kb]) prepared by digesting lambda DNA with EcoRI.

TABLE 2. Frequency of reversion of TnS-induced auxotrophicmutants of P. syringae pv. tomato

Mutant Phenotype Reversion No. of Kms revertants/strain P frequency no. of revertants tested

DC3238 Gln- 1o-10 1/1DC3281 Met- 10-9 20/20DC3181 Ade- 10-9 26/26DC3121 Gln- 1O-10 1/1DC3471 IIV- 10-8 60/60DC3515 Arg- i0-9 60/60DC3195 Gln- <1o-100 a

DC3381 11V- 10-8 38/38DC3420 PyrA- 1O-10 58/58

a-, No revertants were recovered.

Cms transconjugants was obtained by hybridizing blots oftheir digested DNA with labeled probes of the TnS-lackingparental plasmids of pGS9, pCU1, and pACYC184. Nohomologous DNA sequences were observed.

Isolation and characterization of Tn5-induced auxotrophicmutants. Nine (ca. 1%) of 920 Kmr Cms DC3000 transcon-jugants tested were unable to grow on Vogel-Bonner-Kmminimal medium. Analysis of the nutritional requirements ofthese auxotrophs indicated that two (3471 and 3381) wereIlv-, one (3181) was Ade-, one (3281) was Met-, one (3420)was PyrA- (requiring uracil as well as the amino acidarginine), one (3515) was Arg-, and three (3238, 3121, and3195) were Gln- (all requiring a high concentration, 20 mM,of glutamine). Although these results suggest nonrandom-ness for TnS insertion into the P. syringae pv. tomatogenome, Southern blot analysis of the auxotrophs demnon-strated that all nine were the result of unique single-sitemutations (Fig. 1 and 2; data for strains 3515 and 3471 notshown). The Tn5 elements of Gln- mutants 3121 and 3238appeared to be on the same EcoRI, PvuI, and PstI frag-ments. Thus, these mutations, although distinguishable fromeach other, may be closely linked.Spontaneous reversion to prototrophy occurred at low

frequencies (10-8 to 10-10) for eight of the auxotrophs (Table2); no revertants were recovered for strain 3195. Everyrevertant tested was Kms. These results indicate that theauxotrophic phenotype of these mutants is linked to Tn5insertion. Once transposed into the P. syringae pv. tomatogenome, the Tn5 element rarely excises. When it doesexcise, it does not readily move to new sites on the chromo-some.

TABLE 3. P. syringae pv. tomato virulence mutants obtained byTnS mutagenesis

Symptom Hypersensitivity NutritionalStrain expression on reaction on phenotype

tomato leaves tobacco

DC3420 - PyrADC3515 - ArgDC3132 Milda + PrototrophDC3181 + Ade-DC3244 - PrototrophDC3411 - PrototrophDC3456 - PrototrophDC3426 Mild + PrototrophDC3481 + PrototrophaTypical bacterial speck symptoms are shown in Fig. 3. Mild symptom

expression is defined as the production of a very small number of lesions perleaf with or without a reduction in lesion size and chlorotic halo.

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I

A B CFIG. 3. Range of symptoms produced on tomato leaves by

Tn5-induced mutants of P. syringae pv. tomato. (A) Avirulentmutant; (B) mutant with reduced virulence; (C) mutant whichinduced the same symptoms as the parent strain.

Isolation of Tn5-induced virulence mutants. Nine of the 920Kmr CMs transconjugants tested did not produce typicalbacterial speck symptoms on tomato seedlings (Table 3).Seven of the mutants were avirulent (Fig. 3A), while twoproduced mild, barely perceptible symptoms (Fig. 3B).Southern blot analysis of PvuI and EcoRI DNA digests ofthese mutants indicated that they were the result of nineseparate, single-site insertion mutations (Fig. 4) (EcoRl datanot shown). Three of the avirulent mutants (3515, 3420, and3181) were auxotrophic (Table 2). Reversion to prototrophywas accompanied by a return not only to Kms but also tovirulence. These results, together with the Southern blots(Fig. 4, lanes 2 to 4), suggest that, for these three strains, theavirulent and auxotrophic mutant phenotypes are the resultof the same insertion mutation.

P. syringae pv. tomato, like most bacterial plant patho-gens capable of causing plant tissue necrosis, can induce ahypersensitive response in an incompatible host plant (14).The hypersensitive reaction is characterized by rapid plantcell collapse and necrosis followed by localization of thepathogen at the inoculation site. Five of the virulencemutants were unable to induce this reaction on tobaccoleaves.

DISCUSSIONThe yield of Kmr transconjugants in matings between E.

coli WA803(pGS9) and P. syringae pv. tomato was lowerthan that obtained in matings between the same donor and R.meliloti (20) but roughly equivalent to that recorded inconjugation experiments between P. syringae pv.phaseolicola or P. syringae pv. syringae and an E. coli donorcarrying another suicide plasmid vector for TnS, pSUP1011(1). Unlike the pSUP1011-generated P. syringae pv.syringae and pv. phaseolicola transconjugants, which re-tained none of the vector DNA sequences outside of the Tn5region, a small percentage of the P. syringe pv. tomatotransconjugants, being Cmr, did maintain at least a portion ofthe pGS9 vector plasmid. The percentage of Cmr transcon-jugants we found was similar to that reported for R. meliloti.However, as observed with Cmr R. meliloti transconjugants,the pGS9 plasmid did not establish itself as an independentplasmid in P. syringae pv. tomato.The Tn5 element inserted into one unique site on the

chromosome for each of the 14 Tn5-induced P. syringae pv.tomato mutants examined. TnS-induced mutations are rela-

tively stable in this bacterial phytophathogen. Reversionfrequencies (for auxotrophy) were low and neither the TnSelement nor the insertion sequence from Tn5, IS50, readilytransposed to other sites on the genome. These results differfrom those of Anderson and Mills (1), who reported that tworestriction bands hybridized to the labeled Tn5 probe foreach of four of the nine P. syringae pv. syringae andpv.phaseolicola TnS-induced mutants examined. Since thesecond, weaker band did not hybridize to the internalnon-IS50 segment of TnS or to the TnS-lacking parent of thesuicide vector plasmid, they concluded that IS50 had under-gone a secondary transposition in these mutant strains.Meade et al. observed that secondary transposition of Tn5occurs much less frequently in R. meliloti than in E. coli (16).To explain this phenomenon they hypothesized that theTn5-encoded transposition repressor might be more effectivein R. meliloti than in E. coli. Perhaps a similar hypothesiscould be proposed for the differences observed between theP. syringae pv. tomato strain and the closely related P.syringae pv. syringae and pv. phaseolicola strains.The frequency of Tn5-induced auxotrophy reported for

plant-associated bacteria ranges from 0.08% for a slow-growing strain of R. japonicum to 3% for R. meliloti andErwinia carotovora subsp. carotovora strains (1, 3, 16, 19,27). The frequency recorded for P. syringae pv. tomato fellin the middle of this range. The distribution of nutritionalrequirements among P. syringae pv. tomato auxotrophssuggested that Tn5 insertion into the chromosome might notbe totally random. Similar observations were made with theTn5-mutagenized genomes of R. meliloti, E. coli, and P.syringae pv. syringae and pv. phaseolicola (1, 16, 22).However, when DeBruijn and Lupski examined the distri-

1 2 3 4 5 6 7 8 9 10

Kb

_ _ @_~~~~14.3** S _12.7*| 11,94 S. ~ - 9.5

FIG. 4. Southern blot analysis of nine TnS-induced virulencemutants of P. syringae pv. tomato. Blots of PuvI-digested totalcellular DNA were hybridized with 32P-labeled pGS9. Lane 1, StrainDC3000 (wild type); lanes 2 to 10, pathogenicity mutants DC3420,DC3515, DC3181, DC3132, DC3244, DC3411, DC3426, DC3456, andDC3481. The numbers on the right refer to size markers (in kilobases[kb]) prepared by digesting lambda DNA with PvuI.

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bution of TnS insertions in cloned DNA sequences, theyobserved a completely random pattern with no insertionalhot spots (11). Perhaps the nonrandomness seen withmutagenized whole genomes reflects the relative complexityof the biosynthetic pathways and the size of the gene clustersinvolved.

All P. syringae pv. tomato virulence mutants generated byTn5 mutagenesis were the result of a unique single-siteinsertion mutation. However, avirulence for three of thesemutants was linked to auxotrophy (Arg-, Ade-, or PyrA-).A similar observation was made with a TnS-induced methi-onine-requiring mutant of P. syringae pv. phaseolicola (1).Lack of virulence in Arg- and Ade- mutants could be due toan inability to replicate on the nutrient-sparse tomato leafsurface. Why some auxotrophs and not others are affected invirulence could be related to the nutrient composition of theleaf. The PyrA- mutant, however, had a serious metabolicdefect; its generation time in NBY medium was four timesthat of the wild-type parent (Cuppels, unpublished data).Three of the six prototrophic virulence mutants were unableto elicit a hypersensitive response in tobacco leaves. Per-haps these mutants are defective in an early recognitionstep(s) common to both pathogenesis and induction of thehypersensitive reaction.

In summary, Tn5 mutagenesis with suicide plasmid vectorpGS9 is an effective means of generating stable, single-siteinsertion mutations in P. syringae pv. tomato pathogenicitygenes. Using TnS-induced mutants and currently availablemolecular cloning methods (17, 24), we should now be ableto isolate, identify, and characterize these genes and perhapslearn more about their role in the disease process.

ACKNOWLEDGMENTSlam grateful to V.N. Iyerfor supplying plasmids pGS9, pCU1, and

pACY184 and to V. Morris for technical advice on the preparationof Southern blots. The technical assistance of Frederick Smith andCarla Martin is also gratefully acknowledged.

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