A genomic approach to gene fusion technology

Gene expression profiling provides powerful analyses of transcriptional res ponses to cellular perturbation. In contrast to DNA array-based methods, re porter gene technology has been underused for this application. Here we des cribe a genomewide, genome-registered collection of Escherichia coli biolum inescent reporter gene fusions. DNA sequences from plasmid-borne, random fu sions of E. coli chromosomal DNA to a Photorhabdus luminescens luxCDABE rep orter allowed precise mapping of each fusion. The utility of this collectio n covering about 30% of the transcriptional units was tested by analyzing i ndividual fusions representative of heat shock, SOS, OxyR, SoxRS, and cya/c rp stress-responsive regulons. Each fusion strain responded as anticipated to environmental conditions known to activate the corresponding regulatory circuit. Thus, the collection mirrors E. cons transcriptional wiring diagra m. This genomewide collection of gene fusions provides an independent test of results from other gene expression analyses. Accordingly, a DNA microarr ay-based analysis of mitomycin C-treated E. coli indicated elevated express ion of expected and unanticipated genes. Selected loxCDABE fusions correspo nding to these up-regulated genes were used to confirm or contradict the DN A microarray results. The power of partnering gene fusion and DNA microarra y technology to discover promoters and define operons was demonstrated when data from both suggested that a cluster of 20 genes encoding production of type I extracellular polysaccharide in E. coli form a single operon.