Quantitative analysis of bacterial gene expression by using the gusA reporter gene system.
An Azospirillum brasilense Sp7 strain containing a plasmid-borne translational cytN-gusA fusion was grown in a continuous culture to quantitatively evaluate the influence of extracellular signals (such as O(2)) on expression of the cytNOQP operon. The dissolved oxygen concentration was shifted at regular time intervals before the steady state was reached. The measured beta-glucuronidase activity was used to monitor cytN gene expression. However, as the beta-glucuronidase activity in the experimental setup not only depended on altered transcription of the hybrid gene when the signal was varied but was also influenced by cellular accumulation, degradation, and dilution of the hybrid fusion protein, a mathematical method was developed to describe the intrinsic properties of the dynamic bioprocess. After identification and validation of the mathematical model, the apparent specific rate of expression of the fusion, which was independent of the experimental setup, could be deduced from the model and used to quantify gene expression regulated by extracellular environmental signals. In principle, this approach can be generalized to assess the effects of external signals on bacterial gene expression.
Sun
J
J
Centre of Microbial and Plant Genetics, Department of Electrical Engineering, Katholieke Universiteit Leuven, B-3001 Heverlee, Belgium.
Smets
I
I
Bernaerts
K
K
Van Impe
J
J
Vanderleyden
J
J
Marchal
K
K
D015806:Azospirillum brasilense / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D003470:Culture Media; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D005966:Glucuronidase / Q000235:genetics* / Q000378:metabolism; D008954:Models, Biological; D010100:Oxygen / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D003470:Culture Media; D011993:Recombinant Fusion Proteins; C414443:cbb3 oxidase; D003576:Electron Transport Complex IV; D005966:Glucuronidase; D010100:Oxygen