Expression of the indoleacetic acid (iaa) operon, which contributes to the virulence of the phytopathogenic bacterium Pseudomonas syringae subsp. savastanoi, was monitored by using broad-host-range lacZ reporter gene plasmids. A combination of translational (gene) fusions and transcriptional (operon) fusions of P. syringae subsp. savastanoi sequences to lacZ allowed localization of the iaa operon promoter. RNA recovered from P. syringae subsp. savastanoi strains was mapped with iaa operon-specific probes to precisely locate the transcription initiation site. When transcripts from an iaaM::lacZ fusion in Escherichia coli were analyzed, an identical transcription initiation site was observed. The DNA sequence of the iaa operon promoter closely resembled the consensus E. coli promoter sequence. We detected an active, constitutive level of indoleacetic acid biosynthetic gene expression during bacterial growth under a variety of conditions in the absence of host plant influence.The plant pathogenic bacterium Pseudomonas syringae subsp. savastanoi incites the formation of tumorous galls on olive and oleander (40). Tumor formation requires bacterial production and secretion of compounds which act as plant growth hormones, including the auxin indoleacetic acid (IAA) and the cytokinin trans-zeatin (32,36,38). Genes required for IAA and cytokinin biosynthesis in P. syringae subsp. savastanoi have been isolated and sequenced, and homologies with genes present on Agrobacterium tumefaciens tumor-inducing plasmids have been demonstrated (31, 42). Virtually identical IAA biosynthetic genes are present in additional P. syringae subspecies (43). The capacity to synthesize IAA is, in fact, widespread among soil-and plant-associated bacteria (15,25,26). IAA producers include microbes with beneficial effects on plant growth (e.g., Azospirillum species) (2), as well as plant pathogens (15).The IAA biosynthetic pathway of P. syringae subsp. savastanoi can serve multiple functions. In addition to its role in virulence, it can detoxify several tryptophan analogs capable of inhibiting bacterial growth (23). Furthermore, IAA itself can be converted by P. syringae subsp. savastanoi into additional compounds, including an IAA-lysine conjugate (16). P. syringae subsp. savastanoi synthesizes IAA from tryptophan in two steps (22,42). Tryptophan 2-monooxygenase (EC 1.13.12.3), the iaaM gene product, converts L-tryptophan to indoleacetamide. Indoleacetamide hydrolase, the iaaH gene product, catalyzes the conversion of indoleacetamide to IAA. Both iaaM and iaaH are plasmid-borne in P. syringae subsp. savastanoi isolates from oleander hosts, while they have a chromosomal location in strains isolated from olive (6, 9). In each case examined, the * Corresponding author. organization of the region including iaaM and iaaH has been conserved (29). Previous studies indicated that iaaM and iaaH are cotranscribed, with iaaM promoter proximal (8,29).To gain insight into the expression of P. syringae subsp. savastanoi iaa genes, the products of which parti...
The trpE gene, which encodes the large component of the enzyme anthranilate synthase, was isolated from a Pseudomonas syringae subsp. savastanoi (P. savastanoi) cosmid library. Cosmids that complemented an Escherichia coli trpE mutation contained a gene whose product is 86% homologous at the deduced amino acid level to TrpE of Pseudomonas aeruginosa and Pseudomonas putida. Amino acid sequence comparison with other TrpE sequences revealed the existence of conserved regions between the procaryotic and eucaryotic polypeptide sequences analyzed, regions that might be of functional importance. We also report on studies on the expression pattern of this gene. We analyzed the promoter activity of a tipE::lacZ transcriptional fusion, the relative amount of trpE steady-state mRNA, and the activity of anthranilate synthase from cells grown in minimal medium with or without exogenously added tryptophan and in complete medium. We concluded that under the conditions tested, expression of the trpE gene of P. savastanoi is independent of the concentration of tryptophan in the culture medium. Implications of such an expression pattern on the virulence of this bacterium are discussed.
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