Fluorescent proteins can label specific proteins, but those who study anaerobic bacteria could not use these fluorescent proteins because they could not fluoresce without oxygen. Now fluorescent proteins can function without oxygen, thanks to the work of Thomas Drepper and colleagues from Heinrich Heine University Düsseldorf, from EVOcatal GmbH, also of Düsseldorf, and from Research Center Jülich, also in Germany. Others in the group were affiliated with the University of Parma in Italy and with Max Planck Institute for Bioinorganic Chemistry in Mülheim an der Ruhr, Germany. Drepper said that their fluorescent proteins will have numerous applications, including high-throughput screening of drugs that target disease-causing anaerobic bacteria by using fluorescence-activated cell sorting or flow cytometry. Because anaerobic bacteria are being used to detoxify contaminated soils, the fluorescent proteins also could be used to monitor the expression of genes responsible for the cleanup process.These bacterial colonies fluoresce green because they bear the new fluorescent protein reporter derived from Pseudomonas putida.For the basis of the fluorescent proteins, the researchers used two bacterial photoreceptors. The photoreceptors fluoresced weakly, but Drepper and his colleagues knew how to make them fluoresce more robustly because they are similar to well-studied proteins in plants. It was known that the photoreceptors undergo a cycle of fluorescence and darkness when irradiated with blue light. During this cycle, they reversibly bind a flavin mononucleotide chromophore via a cysteine, so the scientists mutated the cysteine to inhibit the cycle and make the photoreceptors fluoresce. More precisely, they mutated the DNA sequence of the receptor protein and inserted the sequence into an inducible reporter system that expressed the flavin mononucleotide-based fluorescent proteins derived from Bacillus subtilis (BsFbFP) and Pseudomonas putida (PpFbFP) photoreceptors. This structural model shows part of a photoreceptor from Bacillus subtilis binding the flavin mononucleotide chromophore (yellow). Researchers modified the flavin mononucleotide-binding domains of this photoreceptor and another photoreceptor from Pseudomonas putida to create novel fluorescent protein reporters that function in anaerobic conditions.Fluorescent signalsNext, the researchers expressed the fluorescent proteins in Escherichia coli and characterized their fluorescence in the presence of oxygen. The bacteria were examined using a Carl Zeiss laser scanning microscope and accompanying software. Both BsFbFP and PpFbFP exhibited 10 times greater fluorescence than the natural photoreceptors. The scientists exploited the evolutionary bias of E. coli to express certain codons to tailor-make BsFbFP specifically for expression in E. coli. The new reporter, called EcFbFP, produced a fluorescent signal 25 times greater than the natural photoreceptors. After this point, the researchers used only EcFbFP and PpFbFP. To determine the dependence of fluorescence on flavin mononucleotide, they used UV-VIS spectroscopy to ascertain the ratio of EcFbFP to flavin mononucleotide, employing a PerkinElmer luminescence spectrometer and a Spex fluorometer. They observed a 1:1 ratio, meaning that flavin mononucleotide does not limit the fluorescence. Fluorimetric measurements also showed that EcFbFP and PpFbFP maximally absorbed at 449 nm and maximally emitted at 495 nm.To measure the quantum yield and photostability, they used a Shimadzu spectrophotometer. The yield measured 0.17 and 0.39 for PpFbFP and EcFbFP, respectively. To determine the photostability, they excited the fluorescent proteins with a blue LED that allowed them to maintain an average light intensity of 2 W/cm2. The researchers said that the proteins exhibited excellent photostability. PpFbFP fluoresced for nearly 2000 s, and EcFbFP emission lasted more than 15,000 s. Bacterial colonies carrying the Pseudomonas putida-derived reporter were imaged under full-spectrum light.Finally, they tested the proteins in the absence of oxygen. They expressed EcFbFP and PpFbFP in the facultative anaerobe Rhodobacter capsulatus and observed the fluorescence with the Carl Zeiss microscope. For comparison, they also expressed GFP-derived yellow fluorescent protein. PpFbFP fluoresced brightly in the absence of oxygen, which Drepper said was the most important finding. The researchers observed no appreciable decrease in fluorescence compared with the fluorescence in aerobic conditions. In contrast, the fluorescence of yellow fluorescent protein completely disappeared in the absence of oxygen, as reported in the April issue of Nature Biotechnology. Drepper said that other researchers are exploring strictly anaerobic bacteria as a cancer treatment and that the fluorescent proteins could label these bacteria during these treatments. Tumors typically contain regions of lower oxygen concentrations, so anaerobic bacteria may selectively kill these cancer cells without harming the rest of the body. Drepper’s group plans to improve the proteins in collaboration with EVOcatal GmbH. Because the quantum yields are comparable to those of some GFP variants but are still relatively low, the team would like to further increase the fluorescence. In addition, it wants to use the fluorescent proteins to label target proteins in cells by fusing them to the fluorescent proteins and checking whether both proteins are still functional.