Chromogenic identification of genetic regulatory signals in Bacillus subtilis based on expression of a cloned Pseudomonas gene.

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Abstract
A method to isolate fragments of DNA that promote gene expression in B. subtilis is described. The system is based on production of catechol 2,3-dioxygenase [CatO2ase; catechol:oxygen 2,3-oxidoreductase (decyclizing), EC 1.13.11.2] encoded by the P. putida TOL plasmid gene xylE. The gene was transferred to a B. subtilis/Escherichia coli plasmid vector 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 chromosomal DNA from B. subtilis, B. licheniformis, B. pumilus and E. coli promoted a 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 sequence 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. subtilis, E. coli and P. putida support the hypothesis. The sensitive color assay offers an approach to develop plasmid gene expression vectors for a wide variety of host organisms.