Vol. 149, No. 1JOURNAL OF BACTERIOLOGY, Jan. 1982, p. 40-460021-9193/82/010040-07$02.W0/O

Cloning and Characterization of iaaM, a VirulenceDeterminant of Pseudomonas savastanoi

LUCA COMAI AND TSUNE KOSUGE*Department ofPlant Pathology, University of California, Davis, California 95616

Received 11 May 1981/Accepted 14 August 1981

Genes for indoleacetic acid production (iaaM and iaaH) are necessary for gallinduction by the olive pathogen Pseudomonas savastanoi. In strain 2009 thesedeterminants are borne on plasmid pIAA1. To map and characterize the genes,fragments of pIAA1 generated by EcoRI endonuclease treatment were cloned inEscherichia coli by using plasmid RSF1010 as vector. We isolated a recombinantplasmid encoding iaaM, the locus for tryptophan 2-monooxygenase. This plas-mid, called pLUC1, was characterized by restriction endonuclease hydrolysis. Itcontained a 2.75-kilobase-pair segment of pIAA1. By cloning this segment in theEcoRI site of pBR328 and pBRH3B we showed that efficient expression of iaaMwas dependent on the orientation with respect to the vector promoters, and thusdetermined the direction of transcription. To more finely map iaaM and confirmthe orientation of transcription, plasmid pLUC1 was subjected to transposon Tnlmutagenesis. The promoter-distal end of iaaM was mapped between coordinatesat 1.7 and 2.15 kilobase pairs of the cloned segment.

Pseudomonas syringae pv. savastanoi (P. sa-vastanoi) induces tumorous overgrowths, calledgalls or knots, on olive and oleander plants (24).Bacterial production of the plant hormone in-dole-3-acetic acid (IAA) is a determinant of gallproduction, as mutants lacking in IAA produc-tion (Iaa- mutants) are not capable of inducinggalls (19), but regain tumorigenicity concomi-tantly with IAA production (Comai and Kosuge,unpublished results).Two enzymes are involved in IAA synthesis:

the first, tryptophan 2-monooxygenase (EC1.13.12.3; Trp monooxygenase), catalyzes theconversion of L-tryptophan to indoleacetamide;the second, indoleacetamide hydrolase, cata-lyzes the hydrolysis of indoleacetamide to IAAand ammonia. Expression of these functions isdependent, in strain 2009, on the presence of a52-kilobase-pair (kb) plasmid, called pIAA1 (3).The aim of this study was to map the IAA

genes and characterize their organization andexpression. With the exception of transforma-tion, systems of genetic analysis are not yetavailable in P. savastanoi; thus, molecular clon-ing of genes of interest represents an attractivechoice.We constructed a recombinant plasmid in

Escherichia coli that bears the gene for themonooxygenase. This locus, designated iaaM,was mapped by restriction endonuclease diges-tion and transposon insertions. High expressionof iaaM was dependent on proper orientation ofthe cloned fragment in a transcriptional unit ofthe vector plasmid.

(A preliminary report on this work was givenat the Annual Meeting of the American Societyof Plant Physiologists, Pullman, Washington,August 1980.)

MATERIALS AND METHODSBacterial strain and pamids. P. savastanoi 2009-7,

a derivative of strain 2009 (3), harbors plasmids pIAA1and RSF1010 and two cryptic plasmids, one 58 kb andthe other 41 kb. E. coli SK1592 is F- gal thi Tl endAsbcBIS hsdR4 hsdM+ (12). The characteristics ofplasmid DNA are described in Table 1.Culture of bacteria. P. savastanoi was routinely

cultured in glucose-peptone medium (medium B)which contains (per liter): proteose peptone, 10 g;D-glucose, 15 g; K2HP04-3H20, 2 g; MgSO4-7H2O,0.4 g (pH 7.0). E. coli was cultured on casein hydroly-sate-glucose-yeast extract medium (medium 523) (11)or on minimal A medium (15). For determination ofIAA and indoleacetamide production, both E. coli andP. savastanoi were grown in medium B. Cells forplasmid extraction and enzyme determinations werewashed in 15mM NaCl and stored at -20°C as pellets.Plasnd preparation. Plasmid DNA from E. coli and

P. savastanoi was isolated by a modification of theprocedures ofHansen and Olsen (6) and of Casse et al.(1). Cells were resuspended in a minimal volume of 15mM NaCl and lysed by the addition of 3% sodiumdodecyl sulfate in TEN (10 mM Tris, 10 mM NaCl, 1mM EDTA [pH 12.45]) to 0.5 of the original culturevolume. The lysate was incubated at 50'C for 10 minand readjusted to pH 8 to 9 with 2 M Tris (pH 5.5), andS M NaCl was added to 1 M final concentration. Afterincubation on ice for at least 1 h, the lysate wascentrifuged at 12,000 x g (40C) for 15 min. Thesupernatant fraction was collected and brought to 10%o(wt/vol) polyethylene glycol 6000 by the addition of

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CLONING A PSEUDOMONAS VIRULENCE GENE 41

TABLE 1. Properties of plasmid

Plasmid Phenotype No. of klRSF1010 Str Sua 8.8pLAA1 Iaa+b 52p38 Cryptic 58p27 Cryptic 41RP4 Amp, Tet, Kan, TraC 57pLUC1 Iam+d 11.6pBR328 Amp, Tet, Cm' 5.0pLUC2 Iam+ 7.7pLUC3 Iam+/-f 7.7pBRH3B Amp, Tet 8pLUC11 Iam`' 10.7

a Str, Streptomycin resistance; Su,resistance.

b Iaa+, Indoleacetic acid production.c Amp, Ampicillin resistance; Tet,

resistance; Kan, kanamycin resistance;transfer.

d Iam+, Indoleacetamide production.' Cm, Chloramphenicol resistance.f Iam+'-, Low-level indoleacetamide X

50%o polyethylene glycol in 10 mM Tris (pincubation for at least 1 h on ice, the pprecipitate was collected by centrifugatio4°C, 10 min), the tubes were drained bypaper towels, and the pellets were suwminimal volume of TES buffer (50 mM 130 mM NaCl, 5 mM disodium EDTA)preparations could be further purified btraction and ethanol precipitation (3) andfor restriction digestion studies after rennol by ethyl ether extraction. Highly pursuitable for cloning were obtained by sabove preparations to density gradient cin CsCl-ethidium bromide (3).Plasmids pBRH3B and pBR328 were j

cultures treated with chloramphenicol amycin (170 ,ug/ml), respectively, by thClewell (2).Restrcton endonudease digestion an

DNA. Conditions for restriction endonution and DNA ligation by T4 DNA ligasspecified by the manufacturers.Transformation. E. coli cells were tra

CaCl2 treatment and heat shock as descrdel and Higa (13). Competent cells were,,glycerol-30 mM CaC12 at -70°C and thavwater bath before use.

Determination of IAA and indoleacetation. The Salkowski reagent was used fmetric detection of indole compounds inas previously described (3, 5). Procedur(tion of neutral and acidic indoles and sublayer chromatography have been describ(3).Assay of Trp monooxygenase and in4

hydrolase. Cell pellets stored at -20°Cand suspended in 5% of the original vcmonooxygenase assay buffer (3), and thesion was disrupted with a Biosonic Isonicator at 0°C in six 15-s intervals. Cments were pelleted by centrifugation at 1

DNA 15 min at 4°C, and the supernatant fraction was usedb Reference

as the enzyme preparation. Assays for Trp monooxy-_ Reference genase and indoleacetamide hydrolase were previous-

15 ly described (3).3 Agrose gel electrophoresis. Agarose gel electropho-3 resis of plasmid DNA was carried out on a horizontal3 slab gel apparatus in 0.5% agarose in Tris-borate buffer7 (14). Running conditions were 40 mA and 10 V/cm.

This report Restriction fragments of plasmid DNA were electro-20 phoresed in 0.7% agarose gel at 12 mA and 3 V/cm.

This report Staining and photography of gels were as describedThis report previously (14).

23 Transposon Tnl mutagenesis. To obtain Tnl inser-This report tion in plasmid pLUC1 (see below), plasmid RP4,

which carries Tnl, was introduced in E. colisulfonamide SK1592(pLUC1) by conjugation. The resulting strain,

harboring both pLUC1 and RP4, was grown at 28°Cfor 24 h, the cells were harvested, and plasmid DNA

tetracycline was extracted as described above. The plasmid prepa-Tra, conjugal ration was digested with the restriction endonuclease

XhoI and transformed in SK1592. As RP4 has twoXhoI sites and pLUC1 has none, most ampicillin-resistant transformants harbored pLUC1 derivatives

)roduction. with a Tn) insertion.Materials. EcoRI restriction endonuclease and T4

DNA ligase were from Miles Biochemicals, Elkhart,?H 8.0). After Ind.; other restriction enzymes were from New En-nlasmid DNA gland Biolabs, Beverly, Mass.; nutrient media weren (5,000 x from Difco Laboratories, Detroit, Mich.; agarose Sea-inversion on kem HGT was from Marine Colloids, Rockland,spended in a Maine; CsCl (technical grade) was from KBI, Revere,risTpH 8.0], Pa. All other reagents and chemicals were from Sigma)y

The above Chemical Co., St. Louis, Mo.'y phenol ex-were suitable RSULTSnoval ofphe-REUTrified samples Construction of pLUCl. To clone the IAA,ubjecting the biosynthetic genes, endonuclease-generatedwentrifugation fragments of pIAA1 were ligated to a vector

plasmid and introduced in E. coli by transforma-isolatedfsrm tion. Previous work (3) had indicated that bothLe method of iaaM and iaaH were on this 52-kb plasmid.

Thus, we expected that some of the chimericId ligation of plasmids generated should bear these determi-clease diges- nants. One was found and named pLUC1.e were those The source of DNA for the construction of

pLUC1 was P. savastanoi strain 2009-7 whichmsformed by contained both pIAA1, the putative source ofibed by Man- the IAA genes, and RSF1010, the vector. Diges-wed in an ice- tion with the restriction endonuclease EcoRI

generated at least 27 fragments from the indige-nide produc- nous P. savastanoi plasmids and linear RSF1010or the colori- (Fig. 1). The digested DNA was ligated with T4culture fluids DNA ligase, and the ligation mixture was trans-es for extrac- formed in E. coli SK1592. Transformed cells)sequent thin- were selected for streptomycin resistance (20ed previously n g/ml). Our original strategy included selection

for 5-methyl Trp resistance since in Pseudomo-doleacetamide nas Trp monooxygenase (iaaM) confers resist-eume of Trp ance to this analog (3). However, since SK1592cell suspen- is naturally resistant to 5-methyl Trp, clones

II (Bronwill) expressing the IAA genes had to be identified'ell wall frag- from production of Salkowski-positive materi-15,000 x g for al(s) (5) in culture filtrates. RSF1010 encodes

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42 COMAI AND KOSUGE

FIG. 1. Restriction endonuclease cleavage analysisof plasmid pLUCI. Lanes: A and H, coliphage lambdatreated with HindIII; B, plasmid DNA from P. savas-tanoi 2009-7 treated with EcoRI; C through G, plasmidpLUC1 treated with EcoRI (C), PstI (D), EcoRI plusPstI (E), PstI plus BamHI (F), PstI plus HindIII (G).

resistance to sulfonamide and streptomycin (16),and insertion ofDNA in the EcoRI site does notaffect resistance to streptomycin (17). Thus,transformants were first selected on streptomy-cin (20 ,g/ml) and then screened for productionof indoleacetamide or IAA. Among 300 strepto-mycin-resistant colonies tested, one positivetransformant was found. The Salkowski-positivesubstance was identified as indoleacetamide.Neither SK1592 nor SK1592(RSF1010) pro-duced indoleacetamide under any conditionstested. IAA was not produced by any strain.Enzyme preparations from the Salkowski-posi-tive putative transformant contained Trp mono-oxygenase activity but no indoleacetamide hy-drolase (Table 2). Neither activity was present inSK1592 or SK1592(RSF1010). Thus, it appearsthat the iaaM locus, but not the iaaH locus, hadbeen cloned.

Plasmid pLUC1 was isolated from the trans-formant and characterized by restriction endo-nuclease digestion (Fig. 1). Its length was 11.6kb; EcoRl digestion yielded two fragments: thelarger (8.8 kb). comigrated with linear RSF1010molecules. The other (2.75 kb) comigrated withone of the fragments produced by digestion ofthe plasmids of strain 2009-7 and is designatedEcoRl-M. We have previously (3) identified thedigestion products of plasmid pIAA1 by compar-ing digests of plasmids from strain 2009 with

those from a derivative that was cured of pIAA1by treatment with acridine orange. EcoRI-Mbelongs to pIAA1. Transformation of differentE. coli K-12 strains with pLUC1 resulted inexpression of iaaM and accumulation of indole-acetamide. We concluded that EcoRI-M bearsthe iaaM locus.

Effect of position on iaaM expression. The ob-servation that high levels of iaaM were ex-pressed in SK1592(pLUC1) was inconsistentwith previous reports of poor expression ofPseudomonas genes in E. coli (9). To determinewhether the high expression was due to anintrinsic property of the iaaM locus or wasdependent on its position with respect to thevector's transcription signals, we cloned theEcoRI-M fragnent in two different vectors. Thefirst, pBR328, is a deletion derivative of pBR325(20) and has a suitable EcoRI site in the chloram-phenicol resistance gene. Preparations ofpLUC1 and pBR328 were digested by EcoRIand then mixed in a 1:1 molar ratio of replicons,ligated, and transformed in SK1592. Recombi-nant plasmids conferring resistance to ampicillinand tetracycline but not to chloramphenicolwere isolated and characterized by restrictionendonuclease digestion (Fig. 2). BamHI diges-tion revealed two classes of recombinant plas-mids each bearing EcoRI-M inserted in differentorientations with respect to the vector. Fourtransformants, two for each orientation classwere tested for Trp monooxygenase activity.Those harboring a pBR328-EcoRI-M chimericplasmid called pLUC2, in which EcoRI-M ispositioned with its Sall site proximal to theampicillin resistance gene (see Fig. 4), expressedvery high levels of Trp monooxygenase (Table2). In contrast, cells harboring pLUC3, therecombinant plasmid in which EcoRI-M has theopposite orientation (see Fig. 4), expresssediaaM to very low level (Table 2). Based on thelocation and direction of promoter sequences onRSF1010 and pBR328, we deduced that tran-scription ofEcoRI-M proceeded from SalI to thePstI site. We also determined that the presenceof a transcription signal on the vector oriented

TABLE 2. Expression of Trp monooxygenase

Sp actStrain Plasmid (103)aP. savastanoi pIAA1 8.5

2009E. coli SK1592 pLUC1 8.8

pLUC2 32.6pLUC3 0.4pLUC11 0.35

a Specific activity: units per milligram of protein.One unit is defined as the amount of enzyme thatcatalyzes the uptake of 1 Fmol of 02 per min.

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CLONING A PSEUDOMONAS VIRULENCE GENE 43

FIG. 2. BamHI restriction endonuclease cleavageanalysis of plasmid pLUC2 (B) and pLUC3 (C); (A)

coliphage lambda treated with HindIII. L, Linear; CC,closed circular.

toward and proximal to the SailI site of EcoRI-M

was necessary for high expression of WaM. To

further prove this point EcoRI-M was cloned in

the EcoRI site of pBRH3B (23). Cloning in that

site inactivates the promoter of the tetracyclineresistance gene which, as a consequence, is not

expressed unless the cloned fragment has a

promoter oriented in the appropriate direction.

A recombinant plasmid, composed of

pBRH3B and EcoRI-M, was constructed bymixing EcoRI-digested pBRH3B and PLUCI,

ligating, and transforming the DNA in SK1592.

Two classes of ampicillin-resistant transfor-

mants were found: those expressing low-level

resistance to tetracycline, as in SK1592(pBRH3B), and those expressing no resistance

to tetracycline. Three plasmids for each pheno-typic class were isolated and characterized byrestriction endonuclease digestion (Fig. 3). Plas-

mids conferring a low level of tetracycline resist-

ance were recircularized pBRH3B. Those that

did not confer tetracycline resistance carried the

EcoRI-M fragmnent inserted with its Pstl site

proximal to the tetracycline resistance gene and

were called pLUC11 (Fig. 4). In SK1592(pLUC11) iaaM was expressed at a very lowlevel (Table 2).Tnl insertion mapping. Approximately 1 in 104

pLUC1 plasmids extracted from SK1592(RP4)(pLUC1) carried a Tn) element. These recombi-nants were isolated on the basis of ampicillinresistance and tetracycline sensitivity (seeabove). According to results presented above,iaaM transcription in pLUC1 is dependent onthe sulfonamide resistance promoter ofRSF1010. Transcription of the streptomycinresistance gene is therefore dependent on read-through of the RNA polymerase beyond theEcoRI-M segment, since the genes for sulfon-amide and streptomycin resistance form an op-eron and are transcribed in that order (18). Thus,to isolate Tnl insertions in EcoRI-M we testedthe pLUC1::Tn) collection for resistance tosulfonamide and sensitivity to streptomycin.Plasmids from colonies exhibiting this pheno-type were isolated after determining whetherindoleacetamide was produced. The plasmidDNA was digested with EcoRI and BamHIseparately, and the digestion products were ana-lyzed by agarose gel electrophoresis. An exam-ple is shown in Fig. 5. EcoRI does not cut Tnl;therefore, we could readily determine whetherTnl had inserted in the RSF1010 moiety or inEcoRI-M since the transposon would contribute

0B C D1.e:= :... ..: .:. :. :.. ::_! ....... . . . . ....... ._

: ::__- . . ..__.

_s .. ..._"

__

g*ye sW.: - .... l- I.. .....

:

. .... : !

.

.:

!im

FIG. 3. Restriction endonuclease cleavage analysisof plasmid pLUC11 (B, D, F) and pBRH3B (C, E, G).Lanes: A, plasmids from 2009-7 treated with EcoRI; Band C, treated with EcoRI; D and E, treated with PstI;F and G, treated with EcoRI plus PstI.

Gm'

._* _-

A B .CABC

'I

-..

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44 COMAI AND KOSUGE

KILOBASE PAIRSI l I I

pLUCI

pLUC 2

pLUC 3

pLUC li

I' I""""'1""""Io""""'1"""I'i""""'1"I""'f"""' I0 1 2 3 4 5 6 7

Su Sm

I A Il I1X0 r n:0D tX rr0R O a 3 0 00H- 0 I H

H H H

FIG. 4. Physical map of recombinant plasmids pLUC1, pLUC2, pLUC3, and pLUC11. The plasmids arerepresented in a linear form obtained by opening the molecule at an imaginary site equidistant from the twoEcoRI sites. The boxed region presents P. savastanoi DNA. Sm, Streptomycin resistance; Su, sulfonamideresistance; Ap, ampicillin resistance; Tc, tetracycline resistance; Cm, chloramphenicol resistance.

an increase of 5.3 kb. One BamHI site occursabout 0.6 kb from the Sail-proximal end ofEcoRI-M; another is located 1.4 kb away fromthe beta-lactamase locus-proximal end of Tnl(8). Thus, digestion with BamHI allows determi-nation of the exact location of Tnl on pLUC1, ifthe orientation of Tnl is known. For sites be-tween coordinates 1.25 to 2.8 kb, starting fromthe Sail-proximal end of EcoRI-M, the orienta-tion of Tnl insertions is evident from the size ofBamHI-generated fragments. In this fashion wemapped a number of insertions on EcoRI-M(Fig. 6). On the basis of inactivation of iaaMexpression, these insertions were divided intotwo groups. The first group, which inactivatediaaM, was represented by insertions less than1.7 kb from the right end of EcoRI-M. Thesecond group consisted of two insertions, themore promoter proximal of which was at thecoordinate 2.15 kb of EcoRI-M. We concludedthat the promoter-distal end of the iaaM struc-tural gene was located between coordinates at1.7 and 2.15 kb.

DISCUSSIONWe aimed to determine the molecular nature

of the gene for Trp monooxygenase (iaaM).

Cloning of iaaM in E. coli established that it isborne on pIAA1. Further, its location wasmapped to EcoRI-M of pIAA1 since this seg-ment ofpIAA1 together with RSF1010 constitut-ed plasmid pLUCL. The choice of RSF1010 wasdetermined by its wide host range (16), whichallows the transfer of cloned DNA from E. colito the host of origin. Introduction ofpLUC1 intopIAA1-less strains of P. savastanoi confers in-doleacetamide production and restores viru-lence (Comai and Kosuge, unpublished results);the phytopathogenic properties conferred bythis chimeric plasmid in this bacterium will bedescribed elsewhere.The cloning strategy employed depended

heavily on expression of the cloned gene. Al-though a positive selection was not possible, theenriched source of the gene (plasmid DNA)assured the success of our attempt. The highlevel of expression of iaaM in pLUC1 by E. coliwas unexpected, as Pseudomonas genes usuallyare not efficiently expressed in this bacterium(9). The EcoRI site of RSF1010 is located in atranscriptional unit between the genes for sul-fonamide and streptomycin resistance, the lastof which is promoter distal (18). Nagahari et al.(17) reported that cloning of the Trp operon in

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VOL. 149, 1982

FIG. 5. Restriction endonuclease cleavageof Tn) insertions in plasmid pLUC1. Lanes:phage lambda treated with HindIII; BCl::TnIB2 Tn) insertion at coordinate 2.1EcoRl-M treated with EcoRI; C, pLUC1treated with BamHI; D, pLUC1: :TnJB6 Tnldinate 0.8 kb of EcoRI-M treated with EcpLUCl: :TnJB6 treated with BamHI. Arrowfaint band in E.

that site resulted in its fusion with theamide-streptomycin operon. Therefiseemed possible that a similar event mEtaken place in pLUC1 with iaaM. To dislbetween the above hypothesis and transinitiation from a Pseudomonas promocloned EcoRI-M in pBR328. This pwhich is a deletion derivative of pBR3allows detection of recombinants by ins4inactivation of the chloramphenicol genexperiment confirmed that iaaM expredependent on a vector promoter as onl)the two possible orientations of EcoRI-]good expression. Noticeably, Trp mono(ase activity in E. coli SK1592(pLUCfourfold higher than that in SK1592(pThe extent to which efficiency of transand the respective copy numbers of 1

CLONING A PSEUDOMONAS VIRULENCE GENE 45

plasmids contribute to this difference was notinvestigated. In addition, the orientation ofiaaMtranscription was found to proceed from the SalIsite toward the PstI site of EcoRI-M, since inboth pLUC1 and pLUC2 (see above) the Sallsite is proximal to the promoter. To conclusivelydemonstrate that a promoter in the vector isnecessary for iaaM expression, EcoRI-M wascloned in pBRH3B, a promoter probe plasmid(23). The resulting chimeric plasmid, pLUC11,expresses iaaM very weakly and does not confertetracycline resistance. The low level of Trpmonooxygenase activity found in cells harboringpLUC3 or pLUC11 could be dependent eitheron background transcription from different vec-tor's promoters or on a Pseudomonas promoterpresent on EcoRl-M but weakly recognized byE. coli RNA polymerase. Our data do not distin-guish between these two alternatives.

Recently, Inouye et al. (10) reported thatcloning xylB and xylE of Pseudomonas putidain E. coli resulted in orientation-dependentexpression of these genes.A shortcoming of this experiment is due to the

presence in both pLUC3 and pLUC11 of tran-scription convergent with that of iaaM. In thefirst plasmid, pLUC3, transcription originatesfrom the chloramphenicol promoter; in the sec-ond plasmid, pLUC11, transcription originatesfrom a promoter located at the beginning of the

* tetracycline gene and oriented toward the Mfragment (21). Convergent transcription inter-feres with expression of the genes of interest

analysis (22). We do not know whether and to whatA, coli- extent it affects iaaM expression.

1, pLU- To map the precise location of iaaM on[5 kb of EcoRI-M, we obtained Tnl insertions in pLUC1:TnlB2 that inactivated resistance to streptomycin butcRIo (e not to sulfonamide. Approximately half of these

points at insertions were in EcoRI-M. This confirmed thatiaaM on pLUC1 is promoter proximal withrespect to the streptomycin resistance locus

sulfon- since insertions in this region have a polar effectore, it on the expression of streptomycin resistance.ay have Keeping in mind the orientation of transcription

,. .-

tinguishcriptioniter, welasmid,25 (20),ertionalie. Thisssion is

( one ofM givesoxygen-2) wasLUCl).,criptionthe two

KILOBASE PAIRSIIIW1.1 . w | * W W |0 1

ioaM-Su

-

AmIt I-a n 0. g o

H FH : H 3 a HH I l

H

I I I I I I I I ' I I I I

2 3

iaaMWSm

0 V=TN IH

FIG. 6. Tnl insertion map of the iaaM locus in therecombinant plasmid pLUC1. Tn) insertions underiaaM- bracket inactivate iaaM; those under iaaM+bracket do not. In all shown insertions Tnl is in thesame orientation, the beta-lactamase locus being prox-imal to the sulfonamide locus.

A B C D E

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46 COMAI AND KOSUGE

we mapped the end of the iaaM locus, corre-sponding to the carboxyl-terminal end of thepeptide, in the region of DNA delimited by themost rightward Tnl insertion inactivating iaaM,and the most leftward Tn) insertion not inacti-vating iaaM. This region is in the proximity ofthe PstI site. We do not know the exact size ofTrp monooxygenase. However, a closely relatedprotein, lysine monooxygenase, isolated fromPseudomonasfluorescens (4) has a subunit mo-lecular weight of 60,000. Assignment of a similarsize to Trp monooxygenase would place theiaaM locus approximately spanning the BamHI-to-PstI segment of EcoRJ-M. This leaves amplespace for a promoter sequence, unless iaaM is apromoter-distal locus of an operon. The latter isnot likely since insertion mutagenesis of pIAA1provided evidence for an IAA operon includingiaaM and iaaH (indoleacetamide hydrolase),with the latter promoter distal (Comai and Ko-suge, unpublished results). Also, the high levelof streptomycin resistance expressed by pLUC1suggests that no termination site is presentdownstream from iaaM.

In summary, cloning iaaM provided a tool forconclusively ascertaining the role of this locus invirulence and showed that iaaM is on plasmidpIAA1. High expression could be achieved byplacing the cloned DNA in a transcriptional unitof the vector. We mapped the locus by restric-tion endonuclease digestion and transposon Tnlmutagenesis, determining both the orientationand the approximate (±0.4 kb) location of iaaM.

ACKNOWLEDGMENTSWe thank R. Rodriguez, B. West, and N. Panopoulos for

plasmids and advice, R. Haselkorn for discussion on conver-gent transcription, and J. Hall for photographic assistance.

This research was supported in part by National ScienceFoundation research grant PCM-7693198.

LITERATURE C1TED1. Casse, F., C. Boucher, J. S. Jullot, M. MAchbe, and J.

Denarle. 1979. Identification and characterization of largeplasmids in Rhizobium meliloti using agarose gel electro-phoresis. J. Gen. Microbiol. 113:229-242.

2. Cewell, D. B. 1972. Nature of ColEl plasmid replicationin Escherichia coli in the presence of chloramphenicol. J.Bacteriol. 110:667-676.

3. Comal, L., and T. Kosuge. 1980. Involvement of plasmiddeoxyribonucleic acid in indoleacetic acid synthesis inPseudomonas savastanoi. J. Bacteriol. 143:950-957.

4. Flshner, M. I. S., and V. Msey. 1974. Purification andproperties of L-lysine monooxygenase from Pseudomo-nasfluorescens. J. Biol. Chem. 249:2579-2586.

5. Gordon, S. A., and R. P. Weber. 1951. Colorimetricestimation of indoleacetic acid. Plant Physiol. 26:192-195.

6. Hanen, J. B., and R. H. Olsen. 1978. Isolation of large

bacterial plasmids and characterization of the P2 incom-patibility group plasmids pMG1 and pMGS. J. Bacteriol.135:227-238.

7. Hedges, R. W., and A. E. Jacob. 1974. Transposition ofampicillin resistance from RP4 to other replicons. Mol.Gen. Genet. 132:31-40.

8. Heffdo, F., P. Bedlnge, J. J. Champoux, and S. Falkow.1977. Deletions affecting the transposition of an antibioticresistance gene. Proc. Natl. Acad. Sci. U.S.A. 74:702-706.

9. Holoway, B. W., V. Krlsnaplla, and A. F. Morgan.1979. Chromosomal genetics ofPseudomonas. Microbiol.Rev. 43:73-102.

10. Inoye, S., A. Nakazwa, and T. Nakazawa. 1981. Molecu-lar cloning of TOL genes xylB and xylE in Escherichiacoli. J. Bacteriol. 145:1137-1143.

11. Kado, C. I., M. G. Heskett, and R. A. IAngley. 1972.Studies on Agrobacterium tumefaciens: characterizationof strains lD135 and B6, and analysis of the bacterialchromosome, transfer RNA and ribosomes for tumor-inducing ability. Physiol. Plant Pathol. 2:47-57.

12. Kushner, S. R. 1978. An improved method for transforma-tion of E. coli with ColEl derived plasmids, p. 17-23. InH. W. Boyer and S. Nicosia (ed.), Genetic engineenrng.Elsevier/North-Holland, New York.

13. Mandel, M., and A. ElIlp. 1970. Calcium dependent phageDNA infection. J. Mol. Biol. 53:159-162.

14. Meyers, J. A., D. Sander, L. P. Elwdl, and S. Falkow.1976. Simple agarose gel electrophoretic method for theidentification and characterization of plasmid deoxyribo-nucleic acid. J. Bacteriol. 127:1529-1537.

15. MIller, J. H. 1972. Experiments in molecular genetics.Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.

16. Nagaari, K., and K. S . 1978. RSF1010 plasmidas a potentially useful vector in Pseudomonas species. J.Bacteriol. 133:1527-1529.

17. Nagahrt, K., T. Tanaka, F. Hishnumna, M. Kurada, andK. Sa hl. 1977. Control of tryptophan synthetaseamplified by varying the numbers of composite plasmidsin E. coli cells. Gene 1:141-152.

18. Rubens, C., F. Hefdon, and S. Falkow. 1976. Transposi-tion of a plasmid deoxyribonucleic acid sequence thatmediates ampicillin resistance: independence from hostrec functions and orientation of insertion. J. Bacteriol.128:425-434.

19. Smaidt, M., and T. Kouge. 1978. The role of indole-3-acetic acid accumulation by alpha-methyl tryptophan-resistant mutants of Pseudomonas savastanol in gallformation on oleanders. Physiol. Plant Pathol. 13:203-214.

20. Soberon, X., L. Covarrubia, and F. BDolivar. 1980. Con-struction and characterization of new cloning vehicles;IV. Deletion derivatives of pBR322 and pBR32S. Gene9:287-305.

21. Steuber, D., and H. Bujard. 1980. Orpnization of tran-scriptional signals in plasmids pBR322 and pACYC184.Proc. Natl. Acad. Sci. U.S.A. 78:167-171.

22. Ward, D. F., and N. E. Mumy. 1979. Convergent tran-scription in bacteriophage lambda: interference with geneexpression. J. Mol. Biol. 133:249-266.

23. West, R. W., R. L. Neve, and R. L. R a . 1979.Construction and characterization of E. coli promoter-probe plasmid vectors. I. Clonings of promoter-containingDNA fragments. Gene 7:271-288.

24. Wilson, E. E. 1935. The olive knot disease: its inception,development, and control. Hilgardia 9:231-264.

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