The construction of a nopaline-inducible marker system for rapid detection of Agrobacterium strains

Abstract

An inducible marker system suitable for Agrobacterium tumefaciens was constructed to enable detection and enumeration of specific bacterial cells introduced into soil. A BamHI cassette carrying the catechol 2,3-dipxygenase (C230) gene, tdnC, fused to the nopaline-inducible Agrobacterium promoter Pi 2[noc] was constructed. This cassette was introduced into the broad host range vector pDSK5019 resulting in plasmid pTVNC2. Inducible C230 activity was observed in an A. tumefaciens strain carrying the plasmid pTVNC2 when nopaline was present. Colonies of bacteria tagged with the system could easily be identified by spraying agar plates containing nopaline with catechol, which is converted to the bright yellow compound 2-hydroxymuconic semialdehyde. The inducible tdnC cassette has also been introduced into the BamHI site of the transposon Tn5 carried by the pSUP1011 suicide vector which can be used as a delivery system for the stable introduction of the inducible marker into the chromosome of target cells.