Chromogenic identification of genetic regulatory signals in Bacillus subtilis based on expression of a cloned Pseudomonas gene (heterospecific gene expression/catechol 2,3-dioxygenase/TOL xylE gene/expression vector)

A method to isolate fragments of DNA that pro- mote gene expression in Bacillus subtilis is described. The system is based on production of catechol 2,3-dioxygenase (CatO2ase; cat- echol:oxygen 2,3-oxidoreductase (decyclizing), EC 1.13.11.2) en- coded by the Pseudomonas putida TOL plasmid gene xylE. The gene was transferred to a B. subtilis/Escherichia coli plasmid vec- tor to construct pTG402. Although xylE is functionally expressed in E. coli, CatO2ase is not detected in B. subtilis unless a fragment of DNA capable of promoting gene expression is ligated into a cleavage site on pTG402 upstream from xylE. Fragments of chro- mosomal DNA from B. subtilis, Bacillus licheniformis, Bacillus pumilus, and E. coli are shown to promote xylE gene expression in B. subtilis. The special feature of the system is the method of detection: colonies of cells that express xylE become yellow within seconds after selection plates are sprayed with catechol, a colorless substrate that is converted by CatO2ase to the yellow product, 2- hydroxymuconic semialdehyde. The complete nucleotide se- quence of xylE is presented. Strong complementarity between the ribosome binding site and 16S rRNA suggests that xylE mRNA translation in B. subtilis may commence at the same site as that recognized by P. putida. Identity of CatO2ase produced in B. sub- tilis, E. coli, and P. putida support the hypothesis. Our sensitive color assay offers an approach to develop plasmid gene expression vectors for a wide variety of host organisms.