Vol. 174, No. 9JOURNAL OF BACTERIOLOGY, May 1992, p. 3021-30290021-9193/92/093021-09$02.00/0Copyright © 1992, American Society for Microbiology

Regulation of Tabtoxin Production by the lemA Gene inPseudomonas syringae

TERESE M. BARTA,'t THOMAS G. KINSCHERF,1 AND DAVID K. WILLIS12*Department of Plant Pathology' and Agricultural Research Service, U. S. Department

ofAgriculture,2 University of Wisconsin, Madison, Wisconsin 53706

Received 18 November 1991/Accepted 4 March 1992

Pseudomonas syringae pv. coronafaciens, a pathogen of oats, was mutagenized with TnS to generate mutantsdefective in tabtoxin production. From a screen of 3,400 kanamycin-resistant transconjugants, sevenindependent mutants that do not produce tabtoxin (Tox-) were isolated. Although the Tn5 insertions withinthese seven mutants were linked, they were not located in the previously described tabtoxin biosynthetic regionof P. syringae. Instead, all of the insertions were within the P. syringae pv. coronafaciens lemA gene. The lemAgene is required by strains of P. syringae pv. syringae for pathogenicity on bean plants (Phaseolus vulgaris). Incontrast to the phenotype of a P. syringae pv. syringae lemA mutant, the Tox- mutants of P. syringae pv.coronafaciens were still able to produce necrotic lesions on oat plants (Avena sativa), although without thechlorosis associated with tabtoxin production. Northern (RNA) hybridization experiments indicated that afunctional lemA gene was required for the detection of a transcript produced from the tbLA locus located in thetabtoxin biosynthetic region. Marker exchange mutagenesis of the tblA locus resulted in loss of tabtoxinproduction. Therefore, both the tblA and lemA genes are required for tabtoxin biosynthesis, and the regulationof tabtoxin production by lemA probably occurs at the transcriptional level.

Tabtoxin is one of a variety of toxins produced by differentisolates of the plant pathogen Pseudomonas syringae (forreviews, see references 13 and 59). Strains that producetabtoxin belong to several subspecies (pathovars) of P.syringae, including but not limited to the pathovars tabaciand coronafaciens (10, 39, 47). The active toxic componentof tabtoxin is tabtoxinine-,-lactam (T,BL), which is releasedwhen tabtoxin is cleaved either by zinc-dependent ami-nopeptidases in the periplasm of the bacterium or by en-zymes in the plant cell (15, 34, 57). Although the structure oftabtoxin (50) resembles that of several monobactam antibi-otics (51, 52), its mode of action is quite different. Tabtoxininhibits the enzyme glutamine synthetase (GS), and thisinhibition is reported to be irreversible (9, 33, 46, 55). Theinhibition of GS in plants results in chlorosis that accompa-nies the necrotic lesions caused by the bacteria on leaves(16, 62). The GS of bacteria or fungi is also inhibited by TfL(14, 18, 54).

Neither the biochemical pathway for tabtoxin synthesisnor the mechanism of self-resistance to TIL is well under-stood. Labeled-precursor studies have suggested that aspar-tate, pyruvate, and L-methionine may serve as buildingblocks for synthesis of T,L (36, 58). Suggested mechanismsfor tabtoxin resistance include GS adenylylation (30) anddetoxification of TPL either by specific I-lactamases (31) orby acetylation (2). There is no evidence 'at a second form ofGS resistant to T,3L is produced by i4;- bacterium, sincepurified GS from tabtoxin-producing ba_ eria is sensitive toinactivation by TEL (54). To date, none of the mechanismsdescribed above has been firmly established as the means bywhich the bacteria protect themselves from tabtoxin.

Initial studies conducted in this laboratory on the geneticsof tabtoxin production involved P. syringae BR2R, a causal

* Corresponding author.t Present address: Gray Freshwater Institute, University of Min-

nesota, Navarre, MN 55392.

agent of bean wildfire that is physiologically identical totobacco wildfire strains, except that it is not a pathogen oftobacco (39). This strain, therefore, is not classified into anyparticular pathovar. P. syringae BR2R was mutagenizedwith Tn5 to generate strains defective in tabtoxin produc-tion. Through Tn5 mutagenesis and cloning procedures, thechromosomal region that encodes tabtoxin biosynthesis wasidentified (29). This region is approximately 25 to 30 kb inlength and contains all of the genetic information necessaryfor the production of and resistance to tabtoxin when intro-duced into a tabtoxin-naive (i.e., nonproducing) strain of P.syringae. However, this region becomes deleted from thechromosome of many tabtoxin-producing P. syringae strainsat a high frequency (approximately 3 x 10-3 per CFUplated), thereby creating technical problems in analyzingTn5-induced Tox- mutants (29). Therefore, we decided toundertake the TnS mutagenesis of P. syringae pv. corona-faciens Pc27R. The tabtoxin region in this strain containsseveral DNA fragments that are conserved in other tabtoxin-producing strains, but it does not become deleted from thechromosome at a detectable frequency. In this paper, wereport that, through the genetic analysis of Tox- mutants ofthis strain, we have identified a gene outside the tabtoxinbiosynthetic region that appears to regulate tabtoxin produc-tion at the level of transcription. This regulatory gene, lemA,is required for pathogenicity of P. syringae pv. syringae (61),and functional lemA alleles have been found in all P.syringae strains that we have examined, including those thatdo not produce tabtoxin (40 and the present study). Inaddition, mutagenesis of a gene regulated by the lemA gene(which we have designated tblA) revealed that the tblA geneis required for tabtoxin production.

MATERIALS AND METHODS

Strains, plasmids, and growth conditions. The strains andplasmids used in this study are presented in Table 1. King'smedium B (KB) (28) was used for the growth of P. syringae

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TABLE 1. Bacterial strains and plasmids

Strain or plasmid Relevant characteristicsa Source or reference

E. coliHB101

DH5otMIKE

P. aenrginosa1937E

P. syringaeBR2R0152Cit7Cit7(lemAp,,J::TnS)

P. syingae pv. coronafaciensPc27

Pc27RKW202-KW208KW209

Pc27R(tbl-9::TnS)

KW210, KW214, KW217,KW218

P. syringae pv. garcae5802

P. syringae pv. glycinea

P. syningae pv. striafaciens1515

P. syringae pv. syringaeB728aNPS3136NUVS1

B728a(lemApn,,3::TnS)P. syningae pv. tabaci

Vir78(R)

Pa522

P. syningae pv. zizaniaePsz2a

pGS9pLAFR3pRK7813pRK415pRK415XpRK2013pRTBL823pTOX9

pWT53

pUFOl

pPC20apLEM57

pBTX2

pLEM16

F- recA13 rspL hsdS20 (hsdR hsdM) thi-1 leuB6 proA2 ara-14 lacYlgalK2 xyl-S mtl-1 supE44 X-

F- recAl hsdRI7 endA1 thi-1 gyrA96 relAl supE44 +80 AJacZAM15 A-Wild-type isolate from sewage

Tox+ Toxr Rif; a causal agent of bean wildfireTox+ Toxr; causal agent of wildfire of soybeanRif'; nonpathogenic; originally designated Cit7R1lemAps,, ::TnS derivative of Cit7 generated by marker exchange of the

lemAp,,l::TnS mutation from P. syringae pv. syringae NPS3136 intoCit7

Tox+ Toxr; a causal agent of halo blight of oats

Rif' derivative of Pc27RTox- Toxr Rif' Kanr; independent TnS derivatives of Pc27RTox- Toxr Rif' Kanr; generated by marker exchange of TnS and flankingDNA from KW203 via pUFOl into Pc27R

Tox- Toxr Rif' Kanr; generated by marker exchange of Tn5 and flankingDNA from pTOX9

Tox- Toxr Rif' Spcr; tblA4::Ql generated by marker exchange withpRK53fQ

Tox+ Toxr; a causal agent of bacterial scorch of coffee (Coffea arabica)Race 4 isolate; bacterial blight of soybeans

Tox- Toxs; a causal agent of bacterial stripe of oats

Rif'; isolate from bacterial brown spot of beanRifr Kanr; lemAl::TnS derivative of B728a; nonpathogenicRif' Kanr Spcr; A(recA)9::fQ lemAl::TnS derivative of B728a;nonpathogenic

Rif' Kanr; generated by marker exchange with lemAp.,3::TnS;nonpathogenic

Tox+ Toxr Rifr; a causal agent of wildfire of tobacco

Tox- Toxs; a causal agent of angular leaf spot of tobacco, previouslydesignated "Pseudomonas angulata"

Tox- Toxs; a causal agent of bacterial leaf streak of wild rice (Zizaniaaquatica)

Camr; TnS vectorTetr; cosmid vectorTetr; pRK404 cosmid derivativeTetr; pRK404 derivative containing the pUC19 polylinkerSame as pRK415, but missing the XbaI site in the polylinkerKanr; mobilizing vectorTetr; pRK7813 carrying the tabtoxin biosynthetic regionTetr Kanr; pLAFR3 containing BR2R chromosomal DNA with the

tbl-9::TnS mutationAmpr; pBluescript II KS+ containing the conserved 5.3-kb PvuII fragmentfrom the BR2R tabtoxin biosynthetic cluster

Tetr Kan'; pRK415 carrying the 17.5-kb Asp718 fragment containing TnSfrom KW203

Tetr; pRK7813 cosmid carrying lemAp., from Pc27RTetr; pRK415 containing carrying lemAp,, from Pc27R within a 5.7-kbchromosomal Asp718-HindIII fragment

Tetr; pLAFR3 clone from B728a gene bank, carrying DNA homologous tothe insert of pUFOl and lemApsc

Tetr; pRK415 carrying lemAp,, from B728a within a 16-kb chromosomalHindIII fragment

7

Bethesda Research Laboratories29

M. Mathew, Glaxo Group Re-search, Greenford, UnitedKingdom

29, 39393740

T. Uchytil and R. D. Durbin,University of Wisconsin

This studyThis studyThis study

This study

This study

ICMPbN. T. Keen and D. Kobayashi,

University of California

R. Bowden, University ofWisconsin

616124

This study

K. K. Knoche and R. D. Durbin,University of Wisconsin

W. C. Nesmith, University ofKentucky

5, 6

44492627This study172929

29

This study

This studyThis study

This study

This study

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TABLE 1-Continued

Strain or plasmid Relevant characteristicsa Source or reference

pLEM12 Tetr; pRK415 carrying lemApsb from BR2R within a 12-kb chromosomal This studyAsp718 fragment

pEMH97 Tetr; pLAFR3 containing lemApss in a 9.7-kb HindIII fragment 24pEMH97/ins-37, Tetr; pEMH97 with Tn3-HoHol insertions in lemAp,, 24pEMH97/ins-124

pEMH97/ins-27, Tetr; pEMH97 with Tn3-HoHol insertions flanking lemApss 24pEMH97/ins-180

pEMH38 Tetr; 3.8-kb XhoI subclone containing lemApss from pEMH97 in pLAFR3 24pRK53 Tetr; pRK415X containing the 5.3-kb Asp718-EcoRI fragment from pWT53 This studypRK53l Tetr Spcr tblA1::fQ This study

a Rif, rifampin; Tet, tetracycline; Kan, kanamycin; Spc, spectinomycin; Tox, tabtoxin; r, resistant; s, sensitive; strain names ending with R, spontaneous Rif'derivative.

b ICMP, International Collection of Microorganisms from Plants (Auckland, New Zealand).

strains. Escherichia coli strains were grown in Luria-Bertanimedium (42). Antibiotics were added to media at the follow-ing concentrations for P. syringae: rifampin (RIF), 100,ug/ml; spectinomycin (SPC), 100 ,ug/ml; kanamycin (KAN),10 ,ug/ml; and tetracycline (TET), 10 ,ug/ml. For E. coli,concentrations were as follows: KAN, 50 jig/ml; TET, 30,ug/ml; ampicillin (AMP), 20 ,ug/ml; and chloramphenicol(CAM) 20 ,ug/ml. Pseudomonas strains used for RNA isola-tion were grown in M9 (35) with 0.5% glycerol. The concen-tration of TET was reduced to 2 ,ug/ml in M9 medium. Stocksolutions of RIF, CAM, and TET were made in 100%methanol at 10 mg/ml and stored at 4°C. KAN and SPC weredissolved in water at concentrations of 10 or 50 mg/ml forKAN and 100 mg/ml for SPC. All water solutions were filtersterilized and stored at 4'C.

Mutagenesis with Tn5, bacterial conjugations, and markerexchange procedures. P. syringae pv. coronafaciens Pc27Rwas mutagenized with Tn5 by using the vector pGS9 carriedin E. coli. Conjugations were performed by plate mating aspreviously described (60). Donor and recipient cells weremixed at ratios varying from 1:1 to 1:4 (donor-to-recipientratio [by volume]). Aliquots of 100 to 200 [lI were plated onKB with RIF and KAN to select for KAN-resistant (Kanr)transconjugants. Kanr transconjugants were transferred withsterile toothpicks to KB master plates in a grid of 48 coloniesper plate and were screened for toxin production as well asauxotrophy, as defined by the lack of growth on definedmineral salts medium (56) with glycerol as a carbon source.To rule out the possibility that Kanr was due to maintenanceof the vector, random colonies were tested for resistance toCAM encoded by pGS9.

Triparental conjugations with the mobilizing plasmidpRK2013 were performed as previously described (60).Site-specific introduction of Tn5 mutations (marker ex-change) was accomplished by using a trans-merodiploidintermediate as previously described for the pLAFR3 vector(60). In all cases, we confirmed successful marker exchangemutagenesis by Southern blot hybridization analysis.

Bioassays for tabtoxin and protease production. Productionof and sensitivity to tabtoxin were determined by bioassay asdescribed elsewhere (18, 29) by using plates of either mineralsalts medium or M9 (also with glycerol). Putative tabtoxin-negative (Tox-) mutants were retested for tabtoxin produc-tion after 48 h of growth with aeration in Woolley's broth(63). A 20-,Al aliquot of culture fluid was absorbed into asterile antibiotic disk, and the disk was placed on thestandard bioassay plates containing the E. coli strain MIKE.Protease production was assayed essentially as describedelsewhere (40).

DNA isolation, hybridization, and general procedures. To-tal genomic DNA from P. syringae was isolated on the basisof the method described by Kinscherf et al. (29). Small-scalepreparations of plasmid DNA were performed by the boilingmethod (20). Large-scale preparations of plasmids weredone by alkaline lysis (42) and purified by two cesiumchloride-ethidium bromide gradients. DNA hybridizationswere carried out as described previously (19). Generaltechniques for DNA manipulation and cloning were carriedout according to standard methods (42). Generation oflabeled DNA probes with random oligonucleotide primers(Pharmacia) was carried out by the procedures of Cobianchiand Wilson (11).

Construction of genomic libraries. A genomic library ofstrain Pc27R was constructed according to standard methods(42) by using the cosmid vector pRK7813. Bacterial alkalinephosphatase (Bethesda Research Laboratories) was used todephosphorylate BamHI-digested vector, which was ligatedto insert DNA (partially digested with Sau3A) at a ratio of10:1 (in micrograms). The ligated DNA mixture was pack-aged in vitro by using the Gigapack II lambda packaging kit(Stratagene) and used to transduce E. coli DH5a accordingto the directions provided with the packaging kit. A genomiclibrary of P. syringae pv. syringae B728a was constructedsimilarly with the vector pLAFR3. The construction of agenomic library of BR2R in pRK7813 has been reportedpreviously (29).

Plasmid constructions. The shuttle vector pRK415 wasmodified by removal of the XbaI site via digestion with XbaIand treatment with the Klenow fragment of DNA polymer-ase (42). After religation and transformation of E. coli DH5at,a plasmid that retained the oa-complementation characteristicof pRK415 was identified. The resultant plasmid, pRK415X,was then used to subclone the 5.3-kb insert of pWT53 as anAsp7l8-EcoRI fragment. The resulting plasmid, pRK53, wasdigested with XbaI, which cuts only once within the insert.After the recessed ends of the XbaI-digested plasmid werefilled in with the Klenow fragment, the fl fragment (38) wasligated into this plasmid as a SmaI fragment. The finalconstruct, pRK53fQ, was used in marker exchange experi-ments.RNA isolations and Northern (RNA) blot hybridizations.

Cultures used for RNA isolation were grown to mid- tolate-log phase in KB with RIF (KAN and TET concentra-tions were lowered to 5 ,ug/ml each when used) and subcul-tured into 100 ml of M9 medium. Cells were harvested fromM9 when the optical density at 600 nm reached 0.8 to 0.9(late log phase). RNA was isolated according to the methoddescribed by Zhu and Kaplan (64), except that RIF was

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3024 BARTA ET AL.

omitted from the isolation solution since strains were Rif.RNA was resuspended in 200 to 500 jxl of sterile water, andits concentration was determined spectrophotometrically at260 nm and by the orcinol reaction (41). For Northernanalysis, samples of 10 to 20 jig of RNA were glyoxlated andresolved in 1.2% agarose gels with Tris-borate buffer (42).RNA molecular weight standards were obtained from Boehr-inger Mannheim Biochemicals. RNAs from brome mosaicvirus were also used as size standards (kindly provided byW. DeJong and P. Ahlquist, University of Wisconsin-Mad-ison). Gels were stained with ethidium bromide, photo-graphed, blotted, and hybridized to 32P-labeled probes asdescribed elsewhere (19).

Plant pathogenicity assays. Pathogenicity on oat plants(Avena sativa cv. Centennial) was tested by localized infil-tration of leaves with suspensions of bacteria. Prior toinoculation, bacteria were grown for 2 days on agar plates at28°C. Cells were resuspended in sterile water at a density ofapproximately 108 CFU/ml (A6., 0.1 to 0.2). Leaves of 12-to 21-day-old oat plants were infiltrated with suspensions ofbacteria made by serial 10-fold dilutions of the suspension of108 CFU/ml suspension. Small crosswise nicks were madewith a razor blade on the epidermis of the leaves, and a smallarea (about 1 cm in length) was infiltrated with inoculum byusing a plastic disposable pipet (Sarstedt). The plants weregrown at 20°C with a 12-h photoperiod. Disease symptomsbecame apparent 3 to 7 days after inoculation, depending onthe inoculum concentration. Inoculations of bean leaveswith mutants of P. syringae BR2R were performed essen-tially as described elsewhere (29).

RESULTS

Isolation and characterization of mutants. Approximately3,400 Kanr transconjugants were screened following TnSmutagenesis of P. syringae pv. coronafaciens Pc27R. Thefrequency of auxotrophy within the mating progeny aver-aged about 1%. Seven Tox- mutants were identified bybioassay. Despite the lack of tabtoxin production, all sevenmutants retained resistance to the toxin (data not shown).To determine whether the insertions in the mutants were

linked, total genomic DNA was digested with several en-zymes (KpnI, EcoRI, or EcoRV) that do not cut within Tn5.Hybridization with the internal HindIll fragment of TnS wasobserved in a 17.5-kb KjpnI fragment in all mutants. Inaddition, one of two EcoRI chromosomal fragments thathybridized to TnS was found in all of the mutants. TnSinsertions also appeared to be located within four differentEcoRV fragments (data not shown).

It has been previously reported that tabtoxin production isphenotypically unstable (8, 56) and that deletions of DNAinvolved in tabtoxin production occur in several tabtoxin-producing strains (29). Therefore, it was necessary to estab-lish that the biosynthetic region was not deleted in the Tox-mutants of strain Pc27R. Genomic DNA from all seven ofthese mutants was digested with PvuII, Southern blotted,and hybridized with pRTBL823, a cosmid that contains thecomplete tabtoxin biosynthetic region cloned from P. syrin-gae BR2R (29). We observed no detectable deletions ofDNA fragments homologous to the probe in any of themutants (data not shown). Also, the pattern of hybridizationindicated that none of the PvuII fragments hybridizing to theprobe was interrupted by TnS. These results demonstratedthat the phenotypes of the mutants were not due to anyobvious mutation in the biosynthetic gene cluster.To demonstrate that the TnS insertions caused the Tox-

1 kb

A

KW203

KCW20 KW202

mTTHI

A

~ KW204KW205 j i

KW207FIG. 1. Physical map showing linkage of TnS insertions in the

Tox- mutants within the 17.5-kbAsp7l8 fragment. The approximatelocations of the TnS insertions in KW201 to KW208 were mapped bySouthern blot hybridization of TnS to chromosomal DNA digestedwithAsp718 and HindlIl. The insertions in the independent mutantsKW206 and KW207 were too close to be accurately resolved by thismethod. A, Asp718; H, HindIII; R, EcoRI.

mutant phenotype, the TnS and flanking DNA from one ofthe mutants (KW203) was cloned as a single Asp718 (iso-schizomer of KpnI) fragment of approximately 17.5 kb insize into the broad-host-range shuttle vector pRK415. Theresultant plasmid, pUFOl, was used to reintroduce the TnSinto the parental strain by homologous recombination. Thephenotypes of the marker exchange derivatives were thesame as those of the primary mutants (Tox- Toxr). Thisindicated that the TnS insertion in the primary mutantKW203 caused the phenotype.Linkage of the seven insertions was confirmed when

pUFOl was used as a probe in hybridization experimentswith chromosomal DNA from the seven mutants. A single17.5-kb Asp718 fragment of DNA hybridized to the probe inall mutants, whereas a single restriction fragment, approxi-mately 12 kb in size, hybridized to the probe in DNA of thewild-type strain Pc27R. The latter fragment was smaller thanthe one hybridizing in the mutants by approximately the sizeof TnS, which is 5.8 kb (32). On the basis of hybridization ofthe insert of pUFOl to chromosomal DNA digested withboth Asp718 (which cuts outside TnS) and HindIII (whichcuts within TnS), it was determined that the TnS insertions inthe mutants were clustered within a 2-kb region of theAsp718 fragment (Fig. 1).

Introduction of the tbl-9::Tn5 mutation into strain Pc27R.Since none of the isolated mutants contained a TnS insertionin the tabtoxin biosynthetic region, we confirmed that dis-ruption of this region of the Pc27R chromosome would resultin loss of toxin production. The tbl-9::TnS insertion is in a5.3-kb PvuII fragment (Fig. 2) that is conserved in alltabtoxin-producing strains (29). The tbl-9::TnS mutation wasintroduced by marker exchange into the chromosome ofPc27R with the plasmid pTOX9. As expected, there was nodetectable tabtoxin production by the strain Pc27R(tbl-9::TnS) (data not shown).

Cloning of the wild-type locus and restoration of Tox-mutants. A pRK7813-based genomic library of P. syringaepv. coronafaciens Pc27R was screened for homology to the17.5-kb Asp718 fragment from pUFOl. Clones hybridizingto the probe were conjugated into five of the Tox- mutants,and two overlapping cosmids that were capable of restoringtabtoxin production were identified. From one of the clones,pPC20a, a 5.7-kb HindIII-Asp7l8 fragment was subcloned

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lemA REGULATION OF TABTOXIN PRODUCTION 3025

2kb

E B BA X EBX EA..I I .a I I a a ..

r 53kb

4,

- ' tbl-9::Tn5

P

12 kb

7.5 kb

P

1.4kb 3.9kb

, ,

FIG. 2. Partial restriction map of the tabtoxin biosynthetic re-gion cloned from P. syringae BR2R, showing the location of theconserved 5.3-kb PvuII fragment used in Northern hybridizationexperiments. The site of the tbl-9::TnS mutation (29) is shown. Thisinsertion was introduced into P. syringae pv. coronafaciens strainPc27R through homologous recombination and resulted in loss oftabtoxin production. Site of insertion of the fl fragment within the5.3-kb PvuII fragment is also indicated. E, EcoRI; B, BamHI; A,Asp718; X, XbaI.

A~~~~~~~~~

ma'=

FIG. 3. Southern blot autoradiograph of Asp718-digested chro-mosomal DNA from several pathovars of P. syringae and P.aeruginosa probed with the 5.7-kb insert from pLEM57. Species orpathovar names are listed at the top of the blot. Tabtoxin-producingstrains are marked with the filled circles. The lane marked "angu-lata" refers to the naturally occurring strains of the pathovar tabacithat do not produce tabtoxin. The representative strains for eachpathovar are (left to right) Pc27R, 5802, 1515, Psz2a, Vir78, Pa522,B728a, Psg race 4, and P. aeruginosa 1937E. Arrows correspond toapproximate sizes of fragments.

into the shuttle vector pRK415. This fragment encompassesthe locations of all Tn5 insertions in the Tox- mutants. Theresulting plasmid, pLEM57, was introduced into five of theTox- mutants (KW202 to KW205 and KW208), and tabtoxinproduction was restored in all transconjugants.

Hybridization of the cloned region to other P. syringaestrains. Strains of P. syringae that do not produce tabtoxinlack a large region of DNA that has been shown to carry acluster of genes required for tabtoxin biosynthesis (29).Therefore, we investigated whether the newly identifiedregion involved in tabtoxin production (cloned in pLEM57)was also specific to tabtoxin-producing strains. The 5.7-kbHindIII-Asp718 fragment from pLEM57 was hybridized toAsp718-digested chromosomal DNA from other strains of P.syringae, including strains that do not produce tabtoxin.Hybridization to the probe was observed in all strainsexamined. Representative pathovar strains are shown in Fig.3. In addition, P. syringae strains BR2R and 0152 hybridizedto the probe (data not shown). The region hybridizing to theinsert of pLEM57 appeared to be conserved in size amongstrains of the pathovars coronafaciens, striafaciens, ziza-niae, and garcae. These four pathovars are virtually identi-cal, on the basis of biochemical and physiological tests (3, 6,43). We also detected hybridization to DNA from strains ofPseudomonas aeruginosa (Fig. 3).

Restoration of mutants with the P. syringae pv. syringaelemA gene. Because of the homology observed between theinsert of pUFOI and both Tox+ and Tox- strains of P.syringae, we used this insert as a probe to screen a genomiclibrary from P. syringae pv. syringae B728a. From over1,000 clones screened, six cosmid clones hybridized to theprobe, and these were tested for their ability to restore toxinproduction to the Tox- mutants. One of these cosmids,designated pBTX2, was able to restore tabtoxin productionto the P. syringae pv. coronafaciens Tox- mutants. A 16-kbHindIlI fragment was subcloned from pBTX2 to producepLEM16, which also was able to complement the mutationsthat caused a Tox- phenotype.Unexpectedly, the restriction maps of pBTX2 and

pLEM16 indicated that the inserts of these plasmids were

physically similar to the region of the P. syringae pv.syringae B728a chromosome carrying the lemA gene (datanot shown). The P. syringae pv. syringae B728a lemA gene(which will be designated hereafter lemA4p,) is containedwithin the insert of the plasmid pEMH97 and is required fordisease symptoms on bean, as well as for the in vitroproduction of syringomycin and protease by P. syringae pv.syringae B728a (22, 23, 40, 61). In addition, Southern blotanalysis showed that pLEM57, the restoring subclone fromthe P. syringae pv. coronafaciens genomic library, hybrid-ized to pEMH97 (Fig. 4). Therefore, we tested the le4Ap,4gene, which encodes a putative regulatory protein (24), forits ability to complement the Tox- mutants. The plasmidpEMH97, containing the lemnAp,4 locus from P. syringae pv.syringae B728a, restored toxin production to all seven of theP. syringae pv. coronafaciens Tox- mutants.

00 00

La VA e~Y4W 3

la° N 0

C; X C; CO

8.9 kb -*7.1 kb -

5.9 kb =5.7 kb

3.3 kb

0.7 kb -);-:

FIG. 4. Southern blot autoradiograph of toxin production-restor-ing cosmids and a subclone from one of the cosmids (pLEM57)probed with pEMH97. The enzymes that were used to digest theplasmids are listed above the lanes. Sizes of fragments wereapproximated by using HindIII-digested lambda DNA as size stan-dards (not shown). The subclone pLEM57 contains only the 5.7-kbHindIII-Asp718 fragment from pPC20a.

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To determine whether it was lemA p,, or other genes linkedto lemAp,,, on the insert of pEMH97 that restored tabtoxinproduction, we tested several derivatives of pEMH97 fortheir ability to restore tabtoxin production to KW203. Theplasmids pEMH97/ins-124 and pEMH97/ins-37, which con-tain Tn3-HoHol insertions in the lemA gene, did not com-plement the mutation in KW203, while Tn3-HoHol inser-tions outside the lemA gene (pEMH97/ins-180 and pEMH97/ins-27) were capable of restoring toxin production toKW203. Furthermore, a much smaller subclone of pEMH97that contains a functional lemA p. gene, pEMH38, wascapable of restoring tabtoxin production to KW203. Lastly,a TnS-containing derivative of B728a was constructed bytransplacement with pUFOl. This strain, B728a (lemAp.,3::TnS), had the same phenotype as the P. syringae pv.syringae strain NUVS1, which carries the lemAp,,,::TnSmutation. On the basis of these data, we concluded that thelocus affected in the Tox- mutants of Pc27R was the P.syringae pv. coronafaciens analog of the lemA gene (desig-nated lemA pr). Accordingly, we have designated the muta-tions within the seven Tox- P. syringae pv. coronafaciensmutants lemAp,c2::TnS through lemAp,,8::TnS.

General requirement of the lemA gene for the expression oftabtoxin within P. syringae. We constructed a lemA mutant ofanother tabtoxin-producing strain, P. syringae BR2R, bymarker exchange using the lemAp,,3::TnS mutation con-tained in pUFOl. As was the case with the P. synngae pv.coronafaciens Tox- mutants, BR2R (lemA,ps3::TnS) lost theability to produce tabtoxin but retained resistance to T,L(data not shown). The plasmid pLEM57 restored tabtoxinproduction to this strain. In addition, several cosmids thathybridized with the insert of pUFOl were identified from agenomic library of strain BR2R. pLEM12, a subclone fromone of the cosmids, was able to restore tabtoxin productionto BR2R (lemApsc3::TnS). This subclone is therefore pre-sumed to carry a functional lemA allele from BR2R (desig-nated lemAPSb). Finally, we introduced the tabtoxin biosyn-thetic genes into P. syringae Cit7 (lemAp,s1::TnS) via theplasmid pRTBL823. As shown previously (29), Cit7(pRTBL823) produced tabtoxin as detected by bioassay.However, although Cit7 (lemAps1l1::TnS) carrying pRTBL823acquired resistance to tabtoxin, it did not produce anydetectable level of the toxin.

Effect of the lemA gene on protease production in strainsPc27R and BR2R. Since the lemA gene appears to berequired for protease production in P. syringae pv. syringaeB728a (23, 40), we assayed Pc27R and BR2R as well as lemAmutants of both strains for protease production. StrainBR2R produced proteolytic zones on plates of skim milkmedium after incubation overnight, while the lemA markerexchange mutant BR2R (lemAp.¢3::TnS) produced no de-tectable protease activity under the same conditions. Theplasmid pLEM12 restored proteolytic activity to BR2R(lemAp.3::TnS) (data not shown). The P. syringae pv.coronafaciens strain Pc27R produced zones indicating pro-teolytic activity of milk proteins after several days of incu-bation. The lemA mutant KW203 failed to produce thisactivity. However, if KW203 contained either pLEM57 orpLEM16, the proteolytic activity was restored (data notshown). In order to detect restoration, TET had to beincorporated into the medium to maintain selection for theplasmids. Therefore, the lemA gene appeared to regulateprotease production in strains BR2R and Pc27R, as it does inP. syringae pv. syringae.The lemA gene is involved in regulation of a tabtoxin locus.

As discussed in the accompanying paper, the sequence of

A

23S RNA -*

16SRNA -*.

1kb )

w w

.1 .1Cl) m

0 0

eq eq3 v

B

w-J

u4 N

N N

a a a

23SRNA -*.

16SRNA -

1kb |0.

FIG. 5. Autoradiograph from Northern blot hybridization of the5.3-kb PvuII fragment from the tabtoxin biosynthetic region (seeFig. 2) to total cellular RNA from wild-type tabtoxin-producingstrains, Tox- mutants, and mutants carrying plasmids with the lemAgene. Positions of the 16S and 23S ribosomal RNAs are indicated bythe arrows. Because of the abundance of these RNA species, thereis an absence of background hybridization at these positions. (A)Strain Pc27R and its derivatives. Strain 2480R is in the pathovarstriafaciens and was used as a negative control since this strain lacksthe tabtoxin biosynthetic DNA region. (B) P. syringae BR2R and itslemA mutant, as well as the lemA mutant carrying the BR2R lemAgene.

the lemA gene predicts that it encodes a transmembraneregulatory protein (24). To determine whether the lemA genewas affecting the transcription of tabtoxin biosyntheticgenes, total cellular RNA from Pc27R and the lemA mutantswas probed with DNA from within the biosynthetic genecluster. The probe was a 5.3-kb PvuII fragment (Fig. 3) thatis conserved in all tabtoxin-producing strains examined andis the site of two TnS insertions that abolished tabtoxinproduction (29). Northern blot hybridizations with the 5.3-kbprobe revealed a major transcript of about 1 kb in size thatwas present in Pc27R but was not detectable in the Tox-mutants that were examined (KW203, KW205, and KW208).The transcript was detected, however, if the Tox- mutantscontained plasmids carrying a wild-type lemA gene fromeither Pc27R or B728a but not the plasmid vector alone (Fig.5A). The level of transcript detected in the complementedmutants appeared to be less than that detected in thewild-type strain Pc27R; however, it is difficult to makequantitative statements, given the general instability of pro-karyotic mRNA. The 1-kb transcript was also detected fromcells grown in rich medium (KB), although the level oftranscript appeared to be lower than the level observed fromcells grown in M9 medium.The 1-kb transcript was not detected in the Tox- mutant

BR2R (lemAp.,3::TnS). As expected, BR2R (lemAnLp3::TnS)was able to produce tabtoxin only when complemented witha wild-type lemA gene, and this observation was correlatedwith the ability to detect the 1-kb mRNA (Fig. SB). Thetranscript was more difficult to detect in BR2R and BR2R(lemAp.¢3::TnS/pLEM12) than in Pc27R and its restoredderivatives. We do not know whether the transcript fromstrain BR2R is produced at a lower level or whether it ismore labile to degradation. Although less tabtoxin is de-

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23S RNA

16S RNAA1kb-.

FIG. 6. Autoradiograph of Northern blot hybridization of the5.3-kb PvuII fragment to RNA from representative marker exchangestrains. RNA from the wild-type strain Pc27R was electrophoresedin a nonadjacent lane in the same gel. Arrows indicate the positionsof the 16S and 23S rRNAs.

tected from strain BR2R in bioassays, we have not deter-mined whether there is a correlation between message levelsand the apparent quantity of tabtoxin produced. The resultsdescribed above indicate that the lemA gene appears toregulate the transcription of at least one tabtoxin gene in thebiosynthetic cluster in both P. syningae pv. coronafaciensand P. syringae BR2R.Mutation of the lemA-regulated tabtoxin gene. We sought to

confirm that the lemA-regulated tabtoxin gene was requiredfor tabtoxin production in strain Pc27R. In order to do this,it was first necessary to localize the gene within the 5.3-kbPvuII fragment. By digesting the 5.3-kb PvuII fragment withXbaI, two separate hybridization probes were generated, 1.4and 3.9 kb in size (Fig. 2). Northern blot hybridizationsindicated that both probes hybridized to the 1-kb lemA-regulated transcript (data not shown). Therefore, we con-cluded that the gene spanned the XbaI site within the 5.3-kbPvuII fragment. We have designated this tabtoxin gene tblA.To confirm the location of the tblA gene and to demon-

strate its role in tabtoxin production, the tblA gene wasmutated by insertion of the fl fragment encoding SPCresistance (38). This was accomplished by recombinationalexchange of the insert contained in pRK53fQ into the chro-mosome of Pc27R. The resulting Tets Spcr progeny (KW210,KW214, KW217, and KW218) did not produce tabtoxin asdetermined by bioassay but were still resistant to the toxin(data not shown). Northern blot analysis was performed todetermine whether the tblA transcript was produced in themarker exchange mutants. Total cellular RNA was isolatedfrom two of the marker exchange derivatives and probedwith the 5.3-kb PvuII fragment. The mRNA detected withthe probe appeared to be truncated (Fig. 6). These dataconfirm that we had mutated the gene responsible for thelemA-regulated transcript and that this gene, designatedtblA, was required for detectable levels of tabtoxin produc-tion in strain Pc27R.

Disease phenotypes of mutants. The ability of the Pc27RlenzApsc mutants to cause disease on oat plants was deter-mined by localized infiltration of leaves with suspensions of104 through 107 CFU/ml. Mutants were compared with theparental strain, Pc27R, and with strains of P. syringae pv.striafaciens. Strains in this pathovar are considered to bephysiologically identical to strains in the pathovar corona-faciens, except for the lack of tabtoxin production (43, 53).Inoculation of the parental strain Pc27R at concentrations of104 through 107 CFU/ml resulted in a spreading necrosissurrounded by a chlorotic zone that is associated with

FIG. 7. Results of pathogenicity assays of leaves of oat plants.The strains used are indicated in the figure. The leaves wereinoculated with bacterial suspensions of 106 CFU/ml. Note thelighter colored area (chlorosis) surrounding the necrotic region onthe leaves inoculated with Pc27R and KW203(pLEM57).

tabtoxin production. Under the conditions of our assay, theTox- lemA mutants retained the ability to cause necrosis onoat leaves, but the necrosis did not spread beyond the areainoculated and was devoid of any chlorotic halo (Fig. 7). At104 cells per ml, the mutants produced small single lesionsthat resembled those produced by P. syringae pv. stria-faciens (data not shown). The same results were observedwith Pc27R (tbl-9::TnS). The lemA mutants restored totabtoxin production produced symptoms similar to thoseproduced by the parental strain (Fig. 7), although there wassometimes less spreading and chlorosis at inoculum densitiesat or below 106 CFU/ml. However, there was no differencebetween the restored mutants and the wild-type strain interms of the length of time during which symptoms devel-oped. The observed differences in the degree of chlorosisand spreading may be accounted for by the instability of thepRK404-derived vectors in the absence of antibiotic selec-tion in plants (12).The lemA mutant derivative of strain BR2R was also

tested for the ability to produce disease symptoms on beanplants. This strain, BR2R (lemApAc3::Tn5), like the Tox-strains described previously (29), appeared to be nonpatho-genic under the condition of our assays.

DISCUSSION

We have isolated tabtoxin-deficient mutants of P. syringaepv. coronafaciens that contain TnS insertions in a regulatorygene, lemAp,c. Initially, we had hoped to mutagenize P.syringae pv. coronafaciens Pc27R in order to better definethe tabtoxin biosynthetic region, since this strain, in contrastto several other tabtoxin-producing strains of P. syringae,did not undergo spontaneous deletion of tabtoxin genes at adetectable frequency (3). Analysis of mutations in this strainshould, therefore, not be complicated by instability of thetabtoxin biosynthetic region. After screening 3,400 possiblemutants, we did not isolate any Tox- progeny of Pc27R thatcontained insertions in the biosynthetic region. Surprisingly,we did identify seven independent insertions of TnS withinthe lemApsc gene. Since the frequency of insertion into asingle gene was much higher than expected for randominsertion events (0.2%), we believe that the lemApSc generepresents a TnS "hot spot" in strain Pc27R. Although Tn5

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has been reported to transpose into the chromosome ofseveral gram-negative species of bacteria in a fairly randomfashion (1, 21, 45, 48), hot spots for this transposon havebeen characterized elsewhere (for a review, see reference 4).The lemA gene appeared to regulate tabtoxin production

via the mRNA produced by the tbUA gene, which we haveshown to be required for tabtoxin production. The regulationof the tblA gene may occur at the level of transcription,although we have not ruled out posttranscriptional effectssuch as message stability. Interestingly, mutations in thelemA gene affected production of tabtoxin, but the Tox-mutants retained resistance to tabtoxin. Therefore, the tab-toxin resistance gene and possibly other biosynthetic genesmust be regulated in a manner different from that of tbU.Furthermore, mutation of tblA did not affect tabtoxin resis-tance. At present, the function of the tblA gene is not known.

Unlike P. syringae pv. syringae, in which the lemA genewas first identified, the lemA gene is not required for lesionformation by P. syringae pv. coronafaciens on its host, A.sativa. Instead, the effects of lemA mutations in P. syringaepv. coronafaciens appeared to be limited to changes in theobserved symptoms. In the case of P. syingae BR2R, amutation in the lemA gene abolished pathogenicity as de-fined by our experimental conditions. However, it is impos-sible to separate the potential effect of a lemA mutation onpathogenicity from its effect on tabtoxin production alone,since mutations in the tabtoxin biosynthetic region of thisstrain also result in a loss of all disease symptoms on beanleaves (29).The lemA gene is an important positive regulator of genes

involved in lesion formation and toxin production in P.syringae. As described in the accompanying paper, the DNAsequence of the lemA gene indicates that it belongs to afamily of two-component regulatory genes (24). However,the network of genes under the control of the lemA geneappears to differ significantly among pathovars. In P. syrin-gae pv. syringae, the lemA gene is required for the produc-tion of syringomycin, extracellular protease (25, 40), andpresumably other unidentified genes that are required fordisease. In P. syringae pv. coronafaciens, the lemA generegulated tabtoxin production as well as protease, but it wasnot an absolute requirement for disease. This suggests thatthe adaptation of each bacterium to its particular host resultsin the utilization of different regulatory pathways. In supportof this idea, other work in this laboratory has shown that amutation of the lemA gene in P. syringae pv. phaseolicola(causal agent of halo blight of bean) did not affect eitherlesion formation or the production of phaseolotoxin (40).However, the lemAp,P locus is able to restore tabtoxinproduction to lemA mutants ofP. syringae pv. coronafaciens(3). At present, it has not been determined what genes areregulated by the lemA gene in P. syringae pv. phaseolicola.Also, we do not know whether the lemA gene regulates othergenes directly or indirectly through other factors. Studies areunder way to identify other lemA-regulated genes and todetermine whether specific promoter sequences of the tblAgene are involved in regulation by the lemA gene product.

ACKNOWLEDGMENTS

Our appreciation is extended to Estelle Hrabak for providingclones with the lemA gene from P. syringae pv. syringae and to JohnEvensta for technical assistance with TnS mutagenesis. We alsothank Caitilyn Alien, Estelle Hrabak, and Jessica Rich for helpfulsuggestions on the manuscript.

This work was partially supported by NIH training grant no.

GM07215 (T.M.B.) and USDA competitive grant no. 88-37263-2856(D.K.W.).

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