JOURNAL OF BACTERIOLOGY, May 1993, p. 3208-3212 Vol. 175, No. 100021-9193/93/103208-05$02.00/0Copyright © 1993, American Society for Microbiology

Isolation and Characterization of a New ChromosomalVirulence Gene of Agrobacterium tumefaciens

I GEDE PUTU WIRAWAN, HI WAN KANG, AND MINEO KOJIMA*Research Institute for Biochemical Regulation, Faculty ofAgriculture,

Nagoya University, Chikusa-ku, Nagoya 464-01, JapanReceived 8 December 1992/Accepted 8 March 1993

A mutant (strain B119) of Agrobacterium tumefaciens with a chromosomal mutation was isolated bytransposon (TnS) mutagenesis. The mutant exhibited growth rates on L agar and minimal medium (AB) platessimilar to those of the parent strain (strain A208 harboring a nopaline-type Ti plasmid). The mutant wasavirulent on all host plants tested: Daucus carota, Cucumis sativus, and Kalanchoe diagremontiana. The mutantwas not impaired in attachment ability to carrot cells. The mutant had one insertion of Tn5 in its chromosome.The avirulent phenotype of B119 was shown to be due to the Tn5 insertion in the chromosome by the markerexchange technique. A wild-type target chromosomal segment (3.0 kb) which included the site of mutationwas cloned and sequenced. Two open reading frames, ORF-1 (468 bp) and ORF-2 (995 bp), were identified inthe 3.0-kb DNA segment. Tn5 was inserted in the middle of ORF-2 (acvB gene). Introduction of the acvB geneinto the mutant B119 strain complemented the avirulent phenotype of the strain. Homology search foundno genes homologous to acvB, although it had some similarity to the open reading frame downstream of thevirA gene on the Ti plasmid. Thus, the acvB gene identified in this study seems to be a new chromosomalvirulence gene ofA. tumefaciens.

Agrobacterium tumefaciens harboring a Ti plasmid incitescrown gall tumors on most dicotyledonous and some mono-cotyledonous plants. During infection, the T-DNA region ofthe Ti plasmid is transferred and integrated into the plantgenome, where its expression results in the formation ofcrown gall tumors (26, 28). In addition to the virulence geneson the Ti plasmid, chromosomal genes are also required forthe virulence of A. tumefaciens (20).

Previously, nine chromosomal genes, viz., chvA (7), chvB(7), att (18), pscA (exoC) (24, 25), chvD (27), chvE (12), ros(5), miaA (10), and the gene reported in our previous paper(14), were identified and characterized. Any mutation inchvA, chvB, att, orpscA (exoC) results in the inability of thebacteria to attach to host cells and the consequent loss ofbacterial virulence. The other four genes, namely, chvD,chvE, ros, and miaA, seem to be involved in virulence byinfluencing the expression of the vir genes on the Ti plasmid.The function of the gene analyzed in our previous paperremains to be determined (14).

It is reasonable to assume that Agrobacterium pathogen-esis entails a large assembly of virulence genes expressed inmany stages of infection which are present on the Ti plasmidas well as on the chromosome. Until now, almost allvirulence genes on the Ti plasmid seem to have beenidentified by extensive genetic analysis, although the func-tions of some of them are still obscure. On the other hand,there seem to be more unidentified virulence genes on thechromosome ofA. tumefaciens. We describe here the isola-tion and characterization of a new chromosomal virulencegene ofA. tumefaciens using transposon mutagenesis.A. tumefaciens and Escherichia coli strains and the plas-

mids used in this study are listed in Table 1. TnS wasintroduced into A. tumefaciens (A208) by conjugation fromE. coli harboring a conjugative plasmid, pJB4J1, containing

* Corresponding author.

Tn5, as reported previously (8). The plasmid pJB4J1 in-cludes a gentamicin resistance gene as well as a neomycinresistance gene in TnS. First, transconjugants were selectedon AB minimal medium (3) containing neomycin. The neo-mycin-resistant colonies were then transferred with tooth-picks onto plates of L agar with and without gentamicin toexamine the presence of the pJB4J1 plasmid. About 75% ofthe colonies were gentamicin sensitive, indicating the ab-sence of the pJB4J1 plasmid. Five thousand neomycin-resistant and gentamicin-sensitive transconjugants were as-sayed for virulence on carrot root disks as previouslydescribed (14). Of these transconjugants, 15 were avirulentor very attenuated. Total DNA containing both the Tiplasmid and chromosomal DNA was isolated from thoseavirulent mutants by the method of Kado and Liu (13). TheDNA samples were subjected to agarose gel electrophoresis.The gel was first stained with ethidium bromide to detect theTi plasmid and chromosomal DNA. Subsequently, the gelwas Southern blotted onto a Hybond N+ nylon membrane(Amersham) (21) and hybridized with probe 1, which wasprepared by labelling the IS50 segment in TnS by using a[32P]dCTP megaprime DNA labelling system (Amersham).For some mutants, hybridization was detected only with theTi plasmid, while for others it was detected only with thechromosomal DNA. Nine avirulent mutants had a TnSinsertion in the chromosome, while six mutants had it in theTi plasmid. We selected one strain (B119) among the mu-tants with the TnS insertion in the chromosome whichshowed the most distinct avirulent phenotype (Fig. 1).Virulence of strain B119 was also examined on hypocotyls ofCucumis sativus and the stem of Kalanchoe diagremonti-ana. Virulence assays on hypocotyls of Cucumis sativuswere done aseptically according to the method of Matsu-moto et al. (17). Virulence was monitored by the size of galls3 weeks after inoculation. Virulence assays of Kalanchoediagremontiana were done by making puncture wounds onthe stem with a sterile toothpick previously smeared withA.

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TABLE 1. Bacterial strains and plasmids

Organism, strain, Characterization(s) Reference or sourceor plasmida

A. tumefaciensA208 Wild type, nopaline-type pTi, tumorigenic 17B119 TnS insertion in the chromosome derivative of A208, avirulent, Knr This studyA M119 A208 marker exchanged with the TnS insertion segment from B119, Knr Genr Tcr This study

E. coliHB101 F- hsdS20(rB- mB-) recA13 ara-14proAZ lacYI galK2 rpsL20 (Smr) xyl-5 mtl-1 Commercial source

supE44 X-JM109 recAl supE44 endEl hsdR17gyrA96 reL41 thi A(lac-proAB) Commercial sourceDH1 supE44 hsdR17 recAl gyrA96 thi-2 relA1 Commercial source

PlasmidspUC18 Cloning vector, Ampr Commercial sourcepBluescript II SK+ Cloning and sequencing vector, Ampr Commercial sourcepRK290 Broad-host-range cloning vector, Tcr 6pRK2013 Mobilization plasmid, Kn' 6pPH1J1 Eviction plasmid, IncP Genr StrW 11pUCD2 Broad-host-range plasmid vector, Tcr Ampr Knr Spr 4pJB4J1 IncP Genr Strr Muc::TnS 11pBR333::IS50 IS50-associated pBR333 22Charomid 9-36 Cosmid vector, Ampr Commercial sourcepNU1 8.8-kb EcoRI DNA fragment containing TnS insertion from B119 cloned in This study

Charomid 9-36pNU2 3.0-kb EcoRI DNA fragment of the wild-type chromosomal locus of TnS This study

insertion cloned in pUC18pNU3 0.7-kb PstI-BamHI DNA fragment from pNU1 cloned in pUC18 This studypNU4 3.0-kb EcoRI DNA fragment from pNU2 recloned in pBluescript II SK+ This studypNU119 8.8-kb EcoRI DNA fragment from pNU1 recloned in pRK290 This studypKW 3.0-kb EcoRI DNA fragment from pNU2 recloned in pUCD2 This studypKWA1, pKWA2, pNU4-deleted derivatives cloned in pUCD2 This study

and pKWA3a The following antibiotics were used at the indicated concentrations (mg/liter): forA. tumefaciens strains, kanamycin (100), neomycin (100), gentamicin (100),

and tetracycline (5); for E. coli strains, ampicillin (100), kanamycin (50), and tetracycline (10).

tumefaciens. The plants were scored for tumor formationafter 5 weeks. Strain B119 was avirulent on both test plants(data not shown).

Strain B119 exhibited a growth rate similar to that ofparent strain A208 on L agar and AB minimal medium.

pTi

Chhr

1 2FIG. 1. The cellular location of the TnS insertion in the avirulent

mutant B119. Total DNA was prepared from strain B119 andelectrophoresed on an agarose gel. The gel was stained withethidium bromide (lane 1) and blotted onto a nylon membrane,which was hybridized with the [32P]dCTP-labelled IS50 (probe 1)(lane 2). pTi, Ti plasmid; Chr, chromosomal DNA.

Attachment ability of strains B119 and A208 to carrot rootcells was assayed as reported earlier (14). No significantdifference in attachment ability was observed between thetwo strains. The attachment abilities of A208 and B119 were2.6 x 107 cells per cm2 and 2.5 x 107 cells per cm2,respectively. The number of TnS insertions in the chromo-some was determined by two experiments. In the firstexperiment, the chromosomal DNA of strain B119 wasdigested by either EcoRI (has no cleavage site within TnS) orBamHI (has one cleavage site) (23). The digested DNAsamples were subjected to Southern hybridization analysisby using probe 1. Only one band (8.8 kb) was detected in thesample digested with EcoRI, while two bands (3.5 and 2.9kb) were detected in the sample digested with BamHI. In thesecond experiment, the chromosomal DNAs of strains A208and B119 were digested by EcoRI, electrophoresed, andSouthern blotted onto a membrane. The membrane washybridized with probe 2, which was prepared from the DNAsegment flanking the TnS insertion site as described below.This probe hybridized to the 3.0-kb band with A208 and tothe 8.8-kb band with the mutant B119. The difference in sizebetween the two bands was 5.8 kb, which exactly matchesthe size of TnS. From these results, it was concluded that thechromosome of strain B119 contained only one insertion ofTnS.

In order to confirm that the avirulent phenotype of B119was due to the TnS insertion in the chromosome, the B119chromosomal DNA segment containing the TnS insertionwas cloned and used to replace the wild-type DNA in the

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3210 NOTES J. BACTERIOL.

1 CGA GTT CTC CGA AGA AGC CCC CCG CGG GCT GCG CGC CCT GCT CGG TCT GGT 5152 GCT TGC TTC CTC GAC CAT CGC AAT TTT CAG CCT GAT GCG GCC GGT TAC CTT 102

103 CAA GCC CGA CTC CAT TCA GCC TGA GGA CGT AGA ACG GGC CAC CGA CAT CGT 153154 CAT GCG ACA GGA TTC GGC CGA CGC CAA TCT GGT GCG CAT GGG CGA CAA GCA 204205 TGT GAT GTT CTC CGA AAG CGG CAA TGC CTT CAT CAT GTA CGG TAT CCA GGG 255256 CCG CTC GTG GAT CGC CTT TGC CGA CCC CGT CGG CGA CGA AGA GGA TTT TCC 306307 CGA CCT CGT CTG GCA ATT CGT GGA AGC CGC CCG TGG AGC CGG CGC CCG CGC 357358 GCT TTC TAC CAG ATT TCG CCC TTC CTT CTC TCC CAT TGT GCC GAT GCG GGC 408409 CTG CGC GCC TTC AAG CTC GGC GAA CTG GCG TTG GTT GAT CTC ACC GCA TTC 459460 GAA CTG AAG GGC GGC AAG CTT GCG ACC CTT CGC CAG TCG CTG AGC CGC GOC 510511 GCG CGC GAC GGC CTC ACC TTC GAG GTT GTC GAG CAA TCG CAG GTG CCC GAT 561562. ATC ATG GAT GAG TTG CAG CAG GTC TCC GAT GGC TGG CTT GCG CAC CAT AAT 612

ORF:.1 > M D E L 0 0 V S D G N L A H H N613 ACG CGG GAA AAG CGC TTC TCG CTT GGC GCT TTC GAA CCG GAC TAT ATT CTG 663

T R E K R F S L G A F E P D Y I L664 TCG CAA CCC GTC GCC GTG CTG CGC AAG GAT GGA AAA ATC ACC 0CC TTC GCC 714

S 0 P V A V L R K D G K I T A F A715 AAT CTG ATG GTG ACG GAG ACG AAA AAG GAA GCC ACC ATC GAC CTC ATG COC 765

N L H V T E T K K E A T I D M L R766 TTT TCG GCG GAT GCG CGC GCG GCT CGA TGG ATT TCC TCT TCG TCA GCA TCA 816

F S A D A R A A R W I S S S S A S817 TGC AGC ATC TGC GCG AGG CGG GAT ATG AAA GCT TCA ATC TCG GCA TGG CGC 867

C S I C A R R D M K A S I S A W R868 CCA TGT CCG GCA TGT CGA AGC GCG ATG CCG CGC CGG TCT GGG ACC GTA TCG 918

P C P A C R S A M P R R S G T V S919 GCA GCA CGC TGT TCG AGC ACG GCG AAC GTT TCT ACA ACT TCA AGG GAC TTC 969

A A R C S S T A N V S T T S R D F970 GCG CCT TCA AGG CAA AGT TCC ACC CGA AAT GGG AAC CCC GTT ACC TTG CTG 1020

A P S R Q S S T R N G N P V T L L1021 TGC AGA ACG GCG TGA CGC CGC TCT GGC ACT GAT GGA TGC GAC GGT TCT GAT 1071

C R T A1072 CAG CGG CGG TGT CAG AGG AGT GAT CGG AAA ATG ATG AAA CGC AAT CTG ATA 1122

ORF >4M M K R N L I1123 GGG GCA TTC ATT GCC GCT TCG ACG CTA CTC TCC TCG TCT GTC GCC TTT TCC 1173

G A F I A A S T L L S S S V A F S1174 CAG GAT AAG CCG GCC TAT GAA ACC GGT ATG ATC CCG GCC GAC CAC ATC ATG 1224

O D K P A Y E T G M I P A D H I M1225 GTG CCC GAT GGC GAT ATC CAG GCC AGC ATC TTC CTG ATT TCC GAC GCC AAT 1275

V P D G D I 0 A S I F L I S D A N1276 GGC TGG ACG GAG GCC GAC GAG ACC CGC GCC AAG GCG CTT GTC GAA AAA GOC 1326

G W T E A D E T R A K A L V E K G1327 GCC GCC GTC GTC GGC ATC GAC TTC AAG GAA TAT CTO AAG GCA CTC GAA GCC 1377

A A V V G I D F K E Y L K A L E A1378 GAT GAC GAC GAG TGC ATC TAC ATG ATC TCG GAC ATC GAG TCG CTG TOG CAG 1428

D D D E C I Y M I S D I E S L S Q1429 CAG ATC CAG CGC ACC GCC GGC ACC GGC AGC TAC CGC CTG CCG ATC GTO ACC 1479

o I 0 R T A G T G S Y R L P I V T1480 GGC ATC GGC AAG GGC GGC ACG CTT GCC CTC GCC ATG ATC GCA CAA AGC CCC 1530

G I G K G G T L A L A M I A Q S P1531 GTT TCC ACC GTC COC GAG GCG GTG GTG GTG GAT CCG AAG GCG 000 CTG CCC 1581

V S T V R E A V V V D P K A G L P1582 CTG GAA AAG ATT CTC TGC ACT CCG GCA ACC AAG GAC AAG GTG GAC GGT GAA 1632

L K I L C T P A T K D K V D G E1633 ACC*C TAT GGC CTG ACA GAC GGT GCT CTG CCG GCA CCG GTC AGC GTC ATC 1683

T V Y G L T D G A L P A P V S V I1684 TTT ACC CCC GAC GCC GAC CAG AAG GGC CGC GAT CAC GTC AAC GCC CTG GTA 1734

F T P D A D Q K G R D H V N A L V1735 AAA CTG CAT TCC GAC ATC GAG GTG ACC GAC GTT ACC GAC AAG GCG GAC GAG 1785

K L H S D I E V T D V T D K A D E1786 GTG CTG ACC CAG ACC CTT TCC GAC AAG GTC GAC GCG GCT GGA GAC AGC GOC 1836

V L T Q T L S D K V D A A G D S G1837 AAC CCG CTC GGG CTT CCG ATC ACG GTT CTG GAG GCA AAA CCT GTG ATG GAC 1887

N P L G L P I T V L E A K P V M D1888 ACC ATG GCG GTG ATC TAT TCC GOC GAT GGC GGC TGG CGC GAC CTG GAC GAG 1938

T M A V I Y S G D G G W R D L D E1939 GAA GTC GGC AGC GCC TTC AGA AGC AGO GTG TGC CGG TCA TTG GCG TCG ATG 1989

E V G S A F R S R V C R S L A S 41990 CCC TGC GGT ATT TCT GGA AGO AAA AGG A* CGA AAG AGG TGG CGG GCM ATC 2040

P C G I S 0 R K R I R K R I R A I2041 TCG CCC GCA TCA TCG ACA CCT ATC GCA AGG AAT GGA AGO TCA AGA ATG TCG 2091

S P A S S T P I A R N G R S R M S2092 TTC TGA TCG GTT ATT CCT TCG GTG CCG ATA TCA TTC CCG CCA CCT ACA ATC 2142

F2143 TCC TGC CCG ACC GGG TGA AAT CCT CCG TTG CGC AGT TTC GCT GCT CG5OCT 21932194 TTC CAA TGA ATG GGA TTT TGA AAT CTC AGT TCA G0G ATG GCT TGG CGT GGC 22442245 TGG TGA AGG CAA GGG CGG CAA GAC CGT CGA TGA CAT CGC CAA GAT CGA CCC 22952296 CAA GCT GGT OCA ATG CGT TTA TGG CAC CGA GOA GOA AGA TGA AGA TCC CTG 23462347 CCC CGG CCT CAA GGC AAA G0G TGT CGA AAC CAT CGG CAT CGA AGG CGM MCA 23972398 CCA TTT

FIG. 2. Nucleotide sequence of the wild-type target chromo-somal locus of the TnS insertion in strain A208. Two open readingframes, ORF-1 (from nucleotide 565 to 1033; 468 bp) and ORF-2(from nucleotide 1102 to 2097, 995 bp), were identified. Underlinedsequences are the putative SD sequences. The closed arrowheadindicates the Tns5 insertion site.

parent strain A208 by marker exchange. The technique usedwas as follows. Chromosomal DNA from B119 was digestedwith EcoRI. The digested DNA was electrophoresed on an

agarose gel, blotted onto a nylon membrane, and hybridizedwith probe 1. A hybridized band (8.8 kb) was ligated into acosmid vector, Charomid 9-36, packaged in vitro and intro-duced into E. coli (DH1). The ampicillin- and neomycin-resistant colonies were selected. The cloned DNA in thesecolonies was analyzed by Southern hybridization analysiswith probe 1. The cosmid vector with the insertion of theTnS-containing fragment was thus selected and namedpNUl. The insert in pNUl was recloned in a broad-host-

range plasmid vector, pRK290. The resulting plasmid(pNU119) was used to transform E. coli (HB101). Subse-quently, the plasmid, pNU119, was mobilized into the parentstrain A208 by triparental mating (9). The tetracycline- andneomycin-resistant transconjugants grown on AB minimalmedium were selected. The eviction plasmid, pPH1JI, wasmobilized into the resulting strains, and the kanamycin- andgentamicin-resistant colonies were selected on AB medium.A double-crossover marker exchange recombinant (AM119)was thus obtained by screening the kanamycin- and genta-micin-resistant colonies for tetracycline sensitivity. Thechromosomal DNAs from AM119 and B119 were digestedwith EcoRI and characterized by Southern hybridizationanalysis with probe 1. In both samples (AM119 and B119),only one hybridization band of the same size (8.8 kb) wasdetected (data not shown). In addition, AM119 was alsoavirulent on carrot root, as was B119.To locate the TnS insertion site in the chromosome of

strain B119, the DNA fragment containing the boundary partbetween the TnS segment and the flanking DNA was clonedas follows. The pNU1 plasmid was digested with EcoRI togenerate the insert. The insert was digested simultaneouslywith BamHI and PstI. The fragment (0.7 kb) thus generated,which contained a part of IS50 (0.65 kb) and the flankingDNA (50 bp), was cloned into pUC18 (pNU3). The boundarypart between the TnS segment and the flanking DNA seg-ment in this plasmid was sequenced. The site of the Tn5insertion was determined by comparing the sequence withthat of the previously reported sequence of TnS (1) (Fig. 2).Awild-type target locus of the TnS insertion in strain A208

was cloned by the following procedures. The 8.8-kb insertDNA in pNU1 was digested by BamHI and EcoRI simulta-neously, resulting in four fragments with sizes of 3.5, 2.7,1.8, and 0.85 kb. Of these, only the 3.5- and 2.7-kb fragmentshybridized with probe 1. The result indicated that the 1.8-and 0.85-kb fragments were derived from the flanking DNAsegment. The 1.8-kb DNA fragment was labelled with[32P]dCTP and used as probe 2. To recover a wild-type targetlocus for TnS insertion, chromosomal DNA from the parentstrain A208 was digested with EcoRI and electrophoresed onan agarose gel. The gel was blotted onto a nylon membrane.On subsequent hybridization with probe 2, a 3.0-kb bandwas detected. This fragment was ligated to plasmid vectorpUC18 and used to transform E. coli (JM109). After screen-ing the transconjugants with probe 2, a positive clone withinsertion of the 3.0-kb DNA fragment was selected andnamed pNU2.The 3.0-kb insert in pNU2 included two BamHI sites,

which were used to orient the insert in a sequencing plasmid.The 3.0-kb fragment from pNU2 was recloned in pBluescriptII SK' (pNU4) for sequencing. The nested deletions in bothorientations of the insert in pNU4 were constructed by usinga Kilo-sequence deletion kit (Takara). The nucleotide se-quence analysis was conducted by using an ABI 373 DNA

241 287A AGDSGNPLGLPITVLEAKPVMDTMAVIYSGDGGWRDLDEEVGSAFRS

B SNGGEAGVRLPLRVFNAkPAKNTVAIIYSGDAGWQNIDEYIGTYLQT32 78

FIG. 3. Amino acid sequence similarity between the C-terminalregion (241 to 287) of AcvB (A) and the N-terminal region (32 to 78)of the open reading frame downstream of the virA gene on theoctopine-type Ti plasmid (B) (19). Colons indicate identical resi-dues, and periods indicate conservative substitutions.

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NOTES 3211

ApKW

pKW^1

pKWU&2

CORFI ORF2 'omplementationI +

+

pKWt&3

BI I 2 3 kb

FIG. 4. Complementation of avirulent mutant B119 by a wild-type target chromosomal segment and its deletion derivatives. (A)Constructs of various chromosomal DNA segments used to complement B119. Those marked with plus (+) complemented the avirulentphenotype, while the one marked with minus (-) did not. (B) Gall formation on hypocotyls of cucumber seedlings inoculated with eithervarious strains ofA. tumefaciens or water as follows: 1, water; 2, B119; 3, A208; 4 to 7, B119 harboring pKW, pKWA1, pKWA2, or pKWA3,respectively; and 8, B119 harboring the pUCD2 plasmid vector.

sequencer from Applied Biosystem (Fig. 2). The nucleotidesequence analysis was conducted with DNASIS software(Hitachi), and a homology search was done with GENETYXsoftware. Within the sequence, two open reading frames,ORF-1 (from nucleotide 565 to 1033; 468 bp) and ORF-2(from nucleotide 1102 to 2097; 995 bp), were identified. Thesequences AGGTG at nucleotide 552 to 556 and AGGA atnucleotide 1087 to 1091 seem to be the ribosome bindingsites (Shine-Delgarno sequence) for ORF-1 and ORF-2,respectively. TnS was inserted in the middle of ORF-2 (acvBgene), as shown in Fig. 2. A homology search of theGenBank nucleotide and NBRF protein data banks found nohomologous genes or proteins. However, a significant ho-mology of 38% was observed in the amino acid sequencebetween the C-terminal region (241 to 287) of AcvB and theN-terminal region (32 to 78) of the open reading framedownstream of the virA gene on the octopine-type Ti plas-mid, whose function is not known (19) (Fig. 3). The aminoacid sequence deduced from the nucleotide sequence showsthat the hydrophobic and hydrophilic stretches alternatealong the sequence of the acvB protein from the N terminusto the C terminus. The N terminus of the acvB protein showsa characteristic of a signal sequence; i.e., the hydrophobicportion at the N terminus is preceded by a short chargedsegment, Met-Met-Lys-Arg.By using the cloned wild-type target locus, complementa-

tion of strain B119 was examined as follows. A wild-typetarget segment (3.0 kb) in pNU2 was excised with EcoRI andwas cloned in pBluescript II SK+. The nested deletionfragments of the segment were constructed by using KpnIand SacI sites in pBluescript II SK+. The whole segmentand the deleted fragments were recloned in the broad-host-range plasmid vector pUCD2 (4) and introduced into themutant B119 by transformation (16). The virulence of thetransformants was assayed on cucumber (Cucumis sativus)hypocotyls. The transformant (pKW) containing the wholewild-type target DNA segment (3.0 kb) complemented strainB119 and produced big galls on cucumber hypocotyls likethose produced by the parent strain A208 (Fig. 4). Thetransformants containing the ORF-1-deleted DNA (pKWA1

and pKWA2) still complemented strain B119. In contrast,the transformant containing ORF-2-deleted DNA (pKWA3)was not able to complement strain B119. These resultsindicated that ORF-2 (acvB gene) complemented the Tn5insertion in strain B119.

In conclusion, the acvB gene isolated in this study seemsto be a new chromosomal virulence gene ofA. tumefaciens.

Nucleotide sequence accession number. The nucleotidesequence has been assigned accession no. D13800 in theDNA Data Bank of Japan.

We thank C. Sasakawa, C. I. Kado, and E. W. Nester for thegenerous gifts of the pBR333::IS50, pUCD2, and pJB4J1 plasmids,respectively. We are also grateful to S. B. Gelvin for providing thepRK2013, pRK290 and pPH1JI plasmids.

This investigation was supported by a grant from the IshidaFoundation.

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