Construction of promoter probe vectors for Synechocystis sp. PCC 6803 using the light-emitting reporter systems Gfp and LuxAB.
Two promoter probe vectors were constructed for the cyanobacterium Synechocystis sp. strain PCC 6803 using reporter genes, which can be easily detected and quantified in vivo by the ability of their encoded proteins to emit light. The vectors allow the transcriptional fusion of promoter sequences with the gfp and luxAB genes, respectively, and their stable integration into a neutral site of the Synechocystis chromosome. Functionality of these vectors was demonstrated by cloning the promoter of the isiAB operon into both promoter probe vectors and analyzing the stress-dependent emission of light by the obtained reporter strains. As was found before for the isiAB operon, the P(isiAB) reporter gene fusions were induced by iron starvation and high salt stress. Induction rates of mRNA of the wild type operon and the reporter gene fusions were found to be essentially the same, indicating that a promoter fragment containing all necessary regulatory elements has been cloned. However, using the gfp gene a slow increase of protein and fluorescence was found, while the luxAB reporter gene constructs led to a rapid increase in luminescence. The same was found after retransfer of cells back into control media, in which the Gfp protein disappeared slowly, while the LuxAB-based luminescence decreased rapidly. These experiments show that both reporter genes can be used in Synechocystis: the luxAB system seems to be favourable regarding reaction time, while the gfp system has the advantage of being independent from any substrate.
Kunert
A
A
Universität Rostock, FB Biologie, Doberaner Str. 143, D-18051, Rostock, Germany. ak@bio4.uni-rostock.de
Hagemann
M
M
Erdmann
N
N
D000447:Aldehydes / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D000458:Cyanobacteria / Q000235:genetics*; D015342:DNA Probes / Q000138:chemical synthesis*; D017930:Genes, Reporter / Q000235:genetics*; D005822:Genetic Vectors / Q000138:chemical synthesis*; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D013379:Substrate Specificity
D000447:Aldehydes; D015342:DNA Probes; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; C021170:decanaldehyde; D008156:Luciferases