LuxArray, a high-density, genomewide transcription analysis of Escherichia coli using bioluminescent reporter strains.
A sequenced collection of plasmid-borne random fusions of Escherichia coli DNA to a Photorhabdus luminescens luxCDABE reporter was used as a starting point to select a set of 689 nonredundant functional gene fusions. This group, called LuxArray 1.0, represented 27% of the predicted transcriptional units in E. coli. High-density printing of the LuxArray 1.0 reporter strains to membranes on agar plates was used for simultaneous reporter gene assays of gene expression. The cellular response to nalidixic acid perturbation was analyzed using this format. As expected, fusions to promoters of LexA-controlled SOS-responsive genes dinG, dinB, uvrA, and ydjM were found to be upregulated in the presence of nalidixic acid. In addition, six fusions to genes not previously known to be induced by nalidixic acid were also reproducibly upregulated. The responses of two of these, fusions to oraA and yigN, were induced in a LexA-dependent manner by both nalidixic acid and mitomycin C, identifying these as members of the LexA regulon. The responses of the other four were neither induced by mitomycin C nor dependent on lexA function. Thus, the promoters of ycgH, intG, rihC, and a putative operon consisting of lpxA, lpxB, rnhB, and dnaE were not generally DNA damage responsive and represent a more specific response to nalidixic acid. These results demonstrate that cellular arrays of reporter gene fusions are an important alternative to DNA arrays for genomewide transcriptional analyses.
Van Dyk
T K
TK
Central Research and Development Department, DuPont Company, Wilmington, Delaware 19880-0173, USA. Tina.K.Van-Dyk@usa.dupont.com
DeRose
E J
EJ
Gonye
G E
GE
D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D016680:Genome, Bacterial; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D016685:Mitomycin / Q000494:pharmacology; D009268:Nalidixic Acid / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis; D020637:Photorhabdus; D013014:SOS Response, Genetics; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D016685:Mitomycin; D009268:Nalidixic Acid; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein