Isolation and molecular characterization of a stationary phase promoter useful for gene expression in Gordonia

Abstract

Gordonia are gram-positive bacteria belonging to Actinomycetes family with a wide variety of industrial and environmental applications. The genetic toolbox, however, is limited for manipulation of these organisms. In the present study, a new promoter has been isolated from Gordonia sp. IITR 100 and characterized in detail. The promoter was found to be functional in Escherichia coli. The minimal promoter was identified in a 166bp fragment by deletion mapping. The putative -35 and -10 hexamer showed four and five nucleotide matches respectively with the E. coli consensus sequence. Three direct repeats and an imperfect inverted repeat upstream to -35 were found. The isolated promoter was found to be six times stronger than the Pkan promoter observed by cloning lacZ downstream to each of them in a plasmid in E. coli. The β-galactosidase activity was maximum at stationary phase and found to be ~800MU for Gordonia sp. IITR 100 and E. coli. This is the first report of a stationary phase promoter isolated and characterized from Gordonia.

Keywords: Deletion mapping; E. coli; Gordonia; Promoter; Stationary phase.