Molecular characterization and expression analysis of the anthranilate synthase gene of Pseudomonas syringae subsp. savastanoi

Abstract

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.

Effects of Mutations in the Pseudomonas putida miaA Gene: Regulation of the trpE and trpGDC Operons in P. putida by Attenuation

“…Nucleotide sequences upstream of the Pseudomonas trpE and trpG genes. Putative Ϫ35 and Ϫ10 regions, leader peptide initiation (met) and termination (stop) codons, and transcription termination signals (term) were deduced from published sequences (10,13,14). The transcription initiation site (indicated by an arrow in each sequence) was confirmed experimentally for P. putida trpE (Fig.…”

“…Effects of miaA1 on transcription of trpE. Published nucleotide sequences (10,13,14) reveal highly conserved sequences that may encode 14-amino-acid polypeptides upstream of trpE in P. putida, P. aeruginosa, and P. syringae and upstream of trpG in P. putida (Table 2). There is a putative leader peptide, but no methionine initiator codon, upstream of trpG in P. aeruginosa, which suggests loss of the ability to be regulated by attenuation.…”

Amplification of trpEG: adaptation of Buchnera aphidicola to an endosymbiotic association with aphids.

“…Among the possible adaptations are constitutive synthesis of the tryptophan biosynthetic enzymes and modifications of AS so as to overcome the effects of feedback inhibition by tryptophan (27). In studies of directed evolution, mutations to constitutivity are common (28); in addition, in some species the tryptophan biosynthetic pathway appears to be constitutive (20,29 *To whom reprint requests should be addressed.…”