LuxArray, a high-density, genomewide transcription analysis of Escherichia coli using bioluminescent reporter strains

Abstract

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.

FIG. 1

Images of duplicate LuxArray 1.0 cellular bioluminescent reporter arrays. Following the spotting of the E. coli strains containing reporter gene fusions onto membranes on LB agar plates and growth for 6 h, the membranes were moved to LB medium plates (A) or LB medium plates containing 5 μg of nalidixic acid/ml (B). The images were taken as described in Materials and Methods immediately after moving the membrane and subsequently at 2, 4, 6, and 8 h and after overnight incubation. A magnification of the 16 spots in the D-4 primary location from panels A and B is shown in panel C. The spot at the secondary location of row 3, column 2, containing cells with upregulated gene fusion dinB-luxCDABE is boxed.

FIG. 2

Total light production from LuxArray 1.0 over time. The averaged pixel density of each spot on triplicate membranes was summed over the entire array and plotted at each time point for each condition. Squares, control LB plates; diamonds, LB plates containing 5 μg of nalidixic acid/ml.

FIG. 3

Scatter plot of showing the relationship of normalized signal intensity with and without nalidixic acid treatment at the 4-h time point. The data from all signals greater than 1.0 for both treatments are plotted.

FIG. 4

Response of the yigN-luxCDABE gene fusion to nalidixic acid (NA) and mitomycin C (MC) in lexA⁺ and lexA1 host strains. Actively growing cultures in LB medium were mixed with chemicals at time zero, and light production was measured in a Luminoskan Ascent microplate luminometer. (A and C) Plasmid pDEW634, containing the yigN-luxCDABE gene fusion, in E. coli strain DM800. (B and D) Plasmid pDEW634 in E. coli strain DM803. RLU, relative light units.