Ordering tryptophan synthase genes of Pseudomonas aeruginosa by cloning in Escherichia coli

Abstract

The Pseudomonas aeruginosa tryptophan synthase genes, tipA and t7pB, which are induced by their substrate indoleglycerol phosphate, were cloned along with their controlling region into the BamHI site of pBR322 to produce the 10.7megadalton plasmid pZAZ5. Sall partial digestion and ligation yielded a smaller plasmid, pZAZ167, with the chromosomal insert reduced in size from 8.1 to 3.4 megadaltons. Both pZAZ5 and pZAZ167 display Pseudomonas-like regulation of the trpA and trpB genes. Deletion of an EcoRI fragment or a BgI fragment from pZAZ167 yielded plasmids pZAZ168 and pZAZ169; the former expresses trpB but not tipA, and the latter has lost both activities. A deleted form of pZAZ5 designated pZAZ1O1 was obtained by excising a BgllI-BamHI segment and religating the tip gene segment in the opposite orientation. This plasmid expresses t7pA and tipB constitutively. The physical maps of these plasmids establish the gene order: promoter-t7pB-trpA.

The roles of indoleglycerol phosphate and the Trpl protein in the expression oftrpBA fromPseudomonas aeruginosa

“…3C). We estimated the size of the SI nuclease-resistant DNA fragment Figure 4. trpI and trpBA promoters and the TrpI binding sites in the 103 bp intergenic region between the two translational start codons (underlined).…”

“…Recently the trpBA gene pair from P. aeruginosa strain PAC 174, along with its regulatory gene, were transferred to E. coli by the broad host range plasmid R68.44 (3). Subsequently these genes were subcloned into an amplifiable pBR322 vector to facilitate deletion studies and DNA sequencing (4,5,6). The trpI regulatory gene modulating the induction of trpBA is located upstream of the trpBA gene pair (5).…”

The expression of the trpD, trpC and trpBA genes of Streptomyces coelicolor A3(2) is regulated by growth rate and growth phase but not by feedback repression

“…A 75 residue protein, TRAP ( trp RNA‐binding attenuation protein), has been demonstrated to bind specifically to trp leader RNA in a tryptophan‐dependent manner (Otridge and Gollnick, 1993). In P. aeruginosa , the seven trp structural genes are scattered at four chromosomal locations (Manch and Crawford, 1981). The trpE and trpGDC genes are repressed by tryptophan, trpF is constitutive, and the trpBA cluster is induced by the substrate of the gene products.…”