Intracellular generation of superoxide as a by-product of Vibrio harveyi luciferase expressed in Escherichia coli.
Luciferase genes are widely used as reporters of gene expression because of the high sensitivity of chemiluminescence detection and the possibility of monitoring light production in intact cells. We engineered fusions of the Escherichia coli soxS promoter to the luciferase structural genes (luxAB) from Vibrio harveyi. Since soxS transcription is positively triggered by the activated SoxR protein in response to agents such as paraquat that generate intracellular superoxide, we hoped to use this construct as a sensitive reporter of redox stress agents. Although a soxR+ soxS'::luxAB fusion exhibited a paraquat-inducible synthesis of luciferase, a smaller increase was consistently observed even in the absence of known soxRS inducers. This endogenous induction was soxR dependent and was further characterized by introducing a plasmid carrying the luciferase structural genes without the soxS promoter into a strain carrying a soxS'::lacZ fusion in the bacterial chromosome. These cells exhibited increased beta-galactosidase expression as they grew into mid-log phase. This increase was ascribed to luciferase activity because beta-galactosidase induction was suppressed (but not eliminated) when the substrate n-decanal was present in the medium. The soxS'::luxAB plasmid transformed superoxide dismutase-deficient strains very poorly under aerobic conditions but just as efficiently as a control plasmid under anaerobic conditions. The production of hydrogen peroxide, the dismutation product of superoxide anion, was significantly increased in strains carrying bacterial luciferase and maximal in the absence of n-decanal. Taken collectively, these data point to the generation of significant amounts of intracellular superoxide by bacterial luciferase, the possible mechanism of which is discussed. In addition to providing insights into the role of superoxide in the activation of the SoxR protein, these results suggest caution in the interpretation of experiments using luciferase as a reporter of gene expression.
González-Flecha
B
B
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, Massachusetts 02115.
Demple
B
B
D001426:Bacterial Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015870:Gene Expression / Q000502:physiology; D005798:Genes, Bacterial / Q000502:physiology*; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic / Q000235:genetics; D018085:Regulon; D013481:Superoxides / Q000378:metabolism*; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics*; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D013481:Superoxides; C070724:SoxR protein, Bacteria; C070725:SoxS protein, E coli; D008156:Luciferases