Construction of a Tn7- lux system for gene expression studies in Gram-negative bacteria

Abstract

A Tn7- lux system was developed for gene expression studies in Gram − bacteria. The plasmids constructed, pHSK.728 and pHSK.729, have the following features: (1) a promoterless Vibrio fischeri lux operon as a reporter system; (2) multiple cloning sites (MCS) ahead of the lux operon, in opposite orientation for the cloning of promoter fragments; (3) a transcriptional terminator ahead of the MCS and translational stop codons in all reading frames before the translational start of the luxC gene; (4) a streptomycin/spectinomycin-resistance encoding gene as a selection marker; and (5) Tn7 border sequences flanking the above elements, permitting the transposition of lux fusion constructs into bacterial genomes. The system was tested using the Escherichia coli lac promoter as well as the differentially regulated promoters of the avrD gene from Pseudomonas syringae pv. tomato and the pelE gene of Erwinia chrysanthemi EC16. Southern blot analysis showed that all fusion constructs had integrated into the host genomes in a single-copy, site-specific manner. The promoters of the avrD and pelE genes resulted in little or no light production when bacteria were grown in rich culture media, but high levels of induction were observed when the bacteria were grown in plant tissues. These results demonstrated that the T'a7- lux system provided a simple, sensitive assay of promoter activity in Gram − bacteria.