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July 13, 2011

FMN-fluorescent protein expression in transformed P. gingivalisIn the mid 1990s, biology went green.  Green Fluorescent Protein, or GFP, that is.  As Dr. Martin Chalfie, a co-recipient of the 2008 Nobel Prize in Chemistry for his ground-breaking work on GFP, then summarized the coming revolution in green luminescence: 

Several bioluminescent [primitive aquatic animals, such as corals and jellyfish] use a secondary fluorescent protein, the green fluorescent protein (GFP), in an energy transfer reaction to produce green light.  The most studied of these proteins have been the GFPs from the jellyfish Aequorea victoria and the sea pansy Renilla reniformis. . . Recent interest in these molecules has arisen from the cloning of Aequorea gfp . . . and the demonstration that its expression in the absence of other Aequorea proteins results in a fluorescent product. This demonstration indicated that GFP could be used as a marker of gene expression and protein localization in living and fixed tissues.

  • Chalfie M. Green fluorescent protein. Photochem Photobiol. 1995 Oct;62(4):651-6.

By the early 2000s, Chalfie’s “could be used” had become a must have to visualize a variety of cellular processes with breathtaking, glow-in-the-dark clarity.  But among microbiologists, there was a rub.  The GFP protein and its variants required oxygen to work their magic, and that limited greatly their applicability to anaerobic, or oxygen-phobic microorganisms.

For scientists who study the oral biofilm, that can be a big problem.  Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium spp., Peptostreptococcus, Eubacterium – all are anaerobic, and all are associated with chronic periodontitis.  Following these bugs and their metabolic activities in real-time would provide a much-needed tool to track the onset and progression of chronic periodontitis.

P. gingivalis association with the ER networkIn the April issue of the journal PLoS ONE, a team of NIDCR-supported scientists reports that it has the solution.  They demonstrated for the first time in P. gingivalis that a strictly anaerobic micro-organism can be genetically engineered to express a green fluorescent protein probe.  In this case, the probe is an adaptation of the recently discovered oxygen-independent flavin mononucleotide (FMN) fluorescent proteins.  Their natural fluorescence derives not from jellyfish, the original source of GFP, but from blue light-sensing proteins of the bacteria Bacillus subtilis and Pseudomonas putida.

In their initial in vitro experiments, the scientists could directly track green protein-expressing P. gingivalis as it invaded living human gingival epithelial cells.  The scientists could distinguish in which cellular compartments copies of the bacterium colocalized, a microscopy term for an overlap of fluorescent tags that would indicate their close spatial proximity.  The researchers also found the bioengineered bacteria were equally adept as natural P. gingivalis at invading the gingival cells and proliferating within them.  Based on their initial work, the scientists said they are confident the technique will be effective in additional cell types and to track the movement of P. gingivalis and other anaerobes in animal models.

 

  • Choi CH, DeGuzman JV, Lamont RJ, Yilmaz Ö.  Genetic transformation of an obligate anaerobe, P. gingivalis for FMN-green fluorescent protein expression in studying host-microbe interaction.  PLoS ONE 2011 April 15:6(4):e18499.

 

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This page last updated: February 26, 2014