Expression of horizontally transferred gene clusters: activation by promoter-generating mutations

Abstract

The occurrence of promoter-generating mutations allowing the transcription of heterologous genes has been studied in a system based on the plasmid-mediated conjugal transfer of histidine biosynthetic genes from a donor bacterium ( Azospirillum brasilense) into a heterologous Escherichia coli mutant population lacking histidine biosynthetic ability and initially unable to recognize the transcriptional signal of the introgressed gene(s). Under selective stressful conditions, His + revertants accumulated in the E. coli His − culture. The number of His + colonies was dependent on the time of incubation under selective conditions, the strength of selective pressure, and on the crowding of cells plated; moreover, it was independent of the physiological status of the cell (i.e. the growth phase). Sequence analysis of plasmid DNA extracted from E. coli His + revertants revealed that single base substitutions in the region upstream of the A. brasilense his operon resulted in an adjustment of the pre-existing sequence that was rendered similar to the E. coli −10 promoter sequence and transcriptable by the host RNA-polymerase. One particular transition (C→T) was predominant in the His + revertants. Data presented here indicated that the barriers to the expression of horizontally transferred heterologous genes or operons may be overcome in a short time scale and at high frequency, and supported the selfish operon model on the origin and evolution of gene clusters.