Breaking down the microbiology world one bite at a time
Christmas lights: Fluorescent proteins in bacteria.
Fluorescent proteins monitor gene expression and protein localization in live cells. The most common green fluorescent protein (GFP) was isolated from Aequorea victoria, a bioluminescent jellyfish, sometimes called crystal jelly. This GFP protein has been optimized to increase brightness and to have a stable signal inside the cells. This also has led to discovering other proteins with a different color in the light spectra, including fluorescent proteins emitting in the blue and yellow spectrum, and red fluorescent proteins which are in the spectrum of visible light.
Regulating the Christmas lights.
Proteins are produced after the translation of an RNA template obtained by the transcription of a DNA template. One of the most common challenges when fluorescent proteins are used in bacteria is the number of copies of DNA templates (plasmids) containing each bacterium. For example, there can be few or many per cell, making a non-homogeneous bacterial population expressing the fluorescent protein. Imagine a street decorated with Christmas lights. The number and decoration type are different in each house (all of them are pretty!), but each one has its own style and its own turn-on/turn-off system. So, the light source, the energy consumption, and the distribution in each house are different.
In biology, when fluorescent proteins are used to study other proteins in bacteria, it is important to have a constant “source of light” with similar energy consumption and distribution inside each cell that comprises a population. In other words, the same number of DNA copies (plasmids) have a similar fluorescence emission between cells.
In this recent study, the authors studied fluorescence stability in a bacterial population with multiple copies of DNA, and compared it with bacteria containing a single copy of this DNA that produces a more homogeneous quantity of fluorescent proteins in each cell. The authors found that despite the signal being similar among cells, a single copy leads to a lower fluorescent intensity in them.
The authors also evaluated a system to control the signal intensity in the bacterial population. Let’s go back to our example of Christmas lights in a street. We know that every house has a system that turns on and off the lights at various times throughout the day. Moreover, some Christmas lights have different lighting modes; some houses choose a constant mode, and others prefer twinkle lights.
So, how can we control the signal intensity in a population of bacterial cells?
In bacteria, some sequences can modulate the expression and degradation of proteins. The authors evaluated three sequences and compared them with fluorescent proteins without these regulatory sequences. They tested protein degradation sequences that modulate the protein stability and allow a balance between protein degradation and fluorescence detection. This is important because fluorescence proteins are usually used to investigate the localization and temporality of a protein in the cell (when the proteins are present and then degraded in a specific situation). Therefore, having stable fluorescent proteins with a well-controlled “turn on/ turn off” system can be used by other researchers to understand protein localization in bacteria.
Finally, the scientist looked at potential applications after characterizing these fluorescence proteins and defining “a bottom” that controls the system. They analyzed protein localization in Salmonella enterica (S. enterica) since this bacterium is vital for studying pathogenic processes, motility, metabolism, and antibiotic responses. They found that when fluorescent proteins are attached to flagellar proteins of bacteria to study motility, the visualization of proteins is more easily detectable when using blue fluorescent proteins than orange or red fluorescent proteins.
Implementing these fluorescent proteins in this bacterium hence contributes to understanding many biological processes in other pathogens.
Link to the original post: Delgadillo-Guevara, M., Halte, M., Erhardt, M. et al. Fluorescent tools for the standardized work in Gram-negative bacteria. J Biol Eng 18, 25 (2024). https://doi.org/10.1186/s13036-024-00420-9
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