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We report the nucleotide sequences of iaaM and iaaH, the genetic determinants for, respectively, tryptophan 2-monooxygenase and indoleacetamide hydrolase, the enzymes that catalyze the conversion of L-tryptophan to indoleacetic acid in the tumor-forming bacterium Pseudomonas syringae pv. savastanoi. The sequence analysis indicates that the iaaM locus contains an open reading frame encoding 557 amino acids that would comprise a protein with a molecular weight of 61,783; the iaaH locus contains an open reading frame of 455 amino acids that would comprise a protein with a molecular weight of 48,515. Significant amino acid sequence homology was found between the predicted sequence of the tryptophan monooxygenase of P. savastanoi and the deduced product of the T-DNA tms-) gene of the octopine-type plasmid pTiA6NC from Agrobacteium tumefaciens. Strong homology was found in the 25 amino acid sequence in the putative FAD-binding region of tryptophan monooxygenase. Homology was also found in the amino acid sequences representing the central regions of the putative products of iaaH and tms-2 T-DNA. The results suggest a strong similarity in the pathways for indoleacetic acid synthesis encoded by genes in P. savastanoi and in A. tumefaciens T-DNA.The association of the tumor-forming bacterium Pseudomonas syringae pv. savastanoi (P. savastanoi) and its hosts, oleander and olive plants, provides a system for studying the molecular basis of virulence of a bacterium in plants. Tumor formation by these plants is a response to high concentrations of indoleacetic acid (IAA) introduced into infected tissue by the bacterium (1); thus, production of a tumor is used to assess virulence of the bacterium. The bacterium produces IAA from tryptophan, with indoleacetamide as the intermediate. The enzymes involved are tryptophan 2-monooxygenase [L-tryptophan:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.3], which catalyzes the conversion of L-tryptophan to indole-3-acetamide, and indoleacetamide hydrolase, which catalyzes the conversion of indoleacetamide to ammonia and IAA (2).The genes for the two enzymes, iaaM and iaaH, are part of an operon that is borne on a plasmid, pIAA, in oleander strains of the pathogen; in olive strains these genes are on the chromosome. Mutants cured of pIAA are weakly virulent on oleander; when transformed with pIAA,

Insertion ofthe transposon Tn5 into the T-region ofthe octopine Ti plasmid ofAgrobacterium tumefiaciens gives rise to crown gall tumors having altered morphology. Three loci within the T-DNA that control tumor morphology have been detected [Garfinkel, D. J., Simpson, R. B., Ream, L. W., White, F. F., Gordon, M. P. & Nester, E. W. (1981). CeU 27, 14-3-1531. They influence tumor size (tnd), production ofroots (tmr), or production of shoots (tins). Cytokinin and auxin levels in such mutant tumors were examined by HPLC/radioimmunoassay and HPLC/fluorescence assay, respectively. Free indoleacetic acid levels (in pmol/g) were: uninfected tobacco stem tissues, 128; wild-type A348 tumors, 295; tin mutant tumors, 307; tmr mutant tumors, 129; and tns mutant tumors, 70. Average trans-ribosylzieatin levels were correspondingly: 0.97, 48, 40, 0.54, and 1,400 pmol/g. transRibosylzeatin/indoleacetic acid ratios were as high as 24 in shootproducing tumors and as low as 0.003 in root-producing tumors. The evidence strongly suggests that tumor phytohormone levels are determined by genes in the T-DNA.It is known from the work ofseveral laboratories that a segment of DNA, the transferred (T) region of a large plasmid (the Ti plasmid) harbored by the inciting bacterium, Agrobacterium tumefaciens, is integrated into the host genome during crown gall tumorigenesis (1). Although seven T-DNA transcripts have been reported (2,3) in octopine' crown gall tumors, no T-DNAcoded gene product has been identified with certainty other than octopine synthase. Other transcripts are presumably determinative in tumor growth and maintenance, but their functions are unknown (4-6). However, crown gall tumors do grow in culture in the absence of added auxin or cytokinin (7), and recent studies (8)(9)(10) indicate that cytokinin levels are increased in many, but not in all,. tumor lines.The morphology of crown gall tumors is determined by at least three factors: (i) the strain ofA. tumefaciens used to incite the tumor, (ii) the plant host, and (iii) the site ofinoculation on the host (11). The work ofSkoog and Miller (12) established that the morphology of normal tobacco tissue in culture is determined by the cytokinin/auxin ratio in the medium. High and low exogenous cytokinin/auxin ratios lead respectively to shoot and root production. It seems very plausible, therefore, that hormone overproduction and imbalance may be general characteristics also of crown gall tumors.Transpositional inactivation of regions of the T-DNA (4-6) has defined several loci that control tumor morphology. We describe here the application of some new (13-14) analytical techniques to the measurement of hormone levels in tumors that have altered morphologies as a result of Tn5 insertion in the T-DNA. The data indicate clearly that endogenous tumor hormone levels are determined by specific loci in the T-DNA that also control tumor morphology. MATERIALS AND METHODSThe strains used in these experiments have been described in detail (4). All mutants were derived from strain...

The phytopathogen Pseudomonas syringae subsp. savastanoi incites the production of galls on olive and oleander plants. Gall formation is dependent on bacterial production of the phytohormone indoleacetic acid (IAA). The genetic determinants for IAA synthesis are located on a plasmid (pIAA) and are organized in an operon in oleander strains of the bacterium. P. syringae subsp. savastanoi further converts IAA to an amino acid conjugate, 3-inidole-acetyl-r-L-lysine (IAA-lysine). The gene for IAA-lysine synthetase (iaaL) was found on the IAA plasmid by screening pIAA deletion mutants for the ability to convert IAA to IAA-lysine. The iaaL locus was then cloned in the vector pUC8 from a bank of P. syringae subsp. savastanoi EW2009 plasmid DNA to construct recombinant plasmid pLG87. The specific activity of IAA-lysine synthetase in Escherichia coli transformed with pLG87 was 47 times higher than that of the enzyme extract from P. syringae subsp. savastanoi. The direction of transcription of the iaaL gene was determined to be opposite to that of the IAA operon. The location of the iaaL gene on pIAAl was mapped by TnS insertion mutagenesis to a 2.5-kilobasepair fragment 2 kilobase pairs from the IAA operon.Pseudomonas syringae subsp. savastanoi (Smith) Stevens incites a disease of oleander (Nerium oleander L.) and olive (Olea europa L.) that is characterized by tumorous outgrowths on the stems and leaves of infected plants (21,26,27). This development of galls is dependent on bacterial production of the phytohormone indoleacetic acid (IAA) (6, 23). The two enzymes involved in the conversion of tryptophan to IAA are tryptophan monooxygenase, which converts tryptophan to indoleacetamide, and indoleacetamide hydrolase, which catalyzes the conversion of indoleacetamide to IAA (16,17). IAA can be further metabolized to an amino acid conjugate, 3-indole-acetyl-e-L-lysine (IAA-lysine) (12, 13).The genes for tryptophan monooxygenase, iaaM, and indoleacetamide hydrolase, iaaH, are organized in an operon in P. syringae subsp. savastanoi (7,8,28); the iaaM locus is the first gene transcribed. In strains isolated from oleander galls, the IAA operon is located on a plasmid (pIAA) (6, 9). Oleander gall isolates selected for resistance to the tryptophan analog a-methyl tryptophan are often incapable of synthesizing IAA and cannot incite the production of galls when inoculated onto oleander plants (23). These avirulent IAA-mutants fall into two classes when surveyed for their plasmid composition: those that lack the IAA operon, either by the loss of the IAA plasmid or by deletion of the IAA genes, and those that contain insertions in the iaaM gene (6,8,9).Certain isolates of P. syringae subsp. savastanoi convert IAA to 13

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