Post By: Lucia Dalle Ore
Pseudomonas
fluorescens is a gram negative, rod shaped bacterium
that commonly inhabits environments like soil, decaying organic matter, and
your local yogurt manufacturing plant. Unlike its sister strain, P. aeruginosa, which has established
itself as an opportunistic pathogen wreaking havoc in immunocompromised
patients, P. fluorescens is actually
a bacterium that has proved itself to be beneficial.
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Figure 1. P. fluorescens
also gets its name from its naturally occurring green fluorescing protein when the iron concentration in the surrounding environment is depleted |
Not only does P. fluorescens work in tandem with plant rhizoids, but they also have been shown to exhibit various antimicrobial properties. More well known for the discovery of Mupirocin, an antibiotic shown to have promising effectiveness against methicillin-resistant Staphylococcus aureus (MRSA), P. fluorescens has actually been shown to have antimicrobial activity against fungus via a secondary phenolic metabolite known as 2,4-diacetylphloroglucinol (DAPG). While the genetics and biochemistry of DAPG have been extensively studied, the antimicrobial mechanisms of how DAPG stalls fungal growth (fungistasis) are fairly unknown. This antimicrobial activity has been explored in Gleeson, O’Gara, and Morrissey’s paper, The Pseudomonas fluorescens secondary metabolite 2,4 diacetylphloroglucinol impairs mitochondrial function in Saccharomyces cerevisiae, where they studied the activity of DAPG in Saccharomyces cerevisiae, or more commonly known as yeast, and the possible mechanisms of antimicrobial activity.
One possibility they considered was the role of ABC transporters, which in fungi and protists exhibit defense mechanisms to effectively efflux toxic metabolites and other irrelevant compounds, dangerous or not, from the cell. In yeast, there is a more specific class of ABC transporters, known as the PDR subfamily like the proteins Pdr5p and Snq2p, that have been shown to play a role in protection against hundreds of chemically unrelated compounds and toxins, with Pdr5p showing specific activity against plant-derived secondary metabolites. However, when they tested the activity of DAPG against strains of yeast where not only the two proteins Pdr5p and Snq2p, but also their transcriptional regulators, Pdr1p and Pdr3p were mutated, they found that it did not increase the effectiveness of DAPG. Therefore, Gleeson and his team figured out that the ABC transport system was not involved in DAPG’s fungistasis mechanism.
One possibility they considered was the role of ABC transporters, which in fungi and protists exhibit defense mechanisms to effectively efflux toxic metabolites and other irrelevant compounds, dangerous or not, from the cell. In yeast, there is a more specific class of ABC transporters, known as the PDR subfamily like the proteins Pdr5p and Snq2p, that have been shown to play a role in protection against hundreds of chemically unrelated compounds and toxins, with Pdr5p showing specific activity against plant-derived secondary metabolites. However, when they tested the activity of DAPG against strains of yeast where not only the two proteins Pdr5p and Snq2p, but also their transcriptional regulators, Pdr1p and Pdr3p were mutated, they found that it did not increase the effectiveness of DAPG. Therefore, Gleeson and his team figured out that the ABC transport system was not involved in DAPG’s fungistasis mechanism.
However, given DAPG’s widespread
effectiveness against a number of fungi, protists, and other microbes, they
deduced that DAPG must be targeting a more central and crucial function. Then,
when they plated yeast cells that had been treated with DAPG, they found that
they exhibited similar properties to yeast that had lost mitochondrial
function, and were therefore unable to grow on nonfermentable carbon sources.
Pursuing the idea that DAPG could be interfering with mitochondrial function,
they plated cells grown in YPD and then exposed to DAPG and stained with MitoTracker
Green. They discovered that there was a positive response in the untreated
cells, with all cells fluorescing. But when incubated with the DAPG, none of
the cells fluoresced. There was no oxidative stress, as shown by
Dihydrorhodamine 123 test which fluoresces when there is damage due to
oxidation, with hydrogen peroxide functioning as the positive control, showing
that the mitochondria were not damaged by DAPG, as illustrated in figure 3. It
was also shown to be a dose-dependent inhibitor of growth when in the presence
of DAPG, with a greater sensitivity when in minimal media.
Figure 2. Yeast grown on nutrient rich and
poor media are both sensitive to DAPG exposure (a) Yeast grown in nutrient-rich culture, and exposed to varing
amounts of DAPG, with the black circles representing the least amount of
exposure, with gradual increase in exposure represented by the other lines with
the clear triangle representing maximum exposure. b) Yeast grown in nutrient-poor culture and also exposed to varing
amounts of DAPG.
Figure 3. When treated with DAPG, mitochondrial function is severely decreased, but doesn’t cause immediate oxidative stress. (a) Wild type yeast cells were allowed to grow to mid-log phase, then treated with DAPG and stained with MitoTracker Green and viewed using epifluorescence microscopy. (b) Yeast was allowed to grow to mid-log phase and incubated with Dihydrorhodamine 123, then the cultures were split, with one half being treated with H2O2, and the other exposed to DAPG. The cells were then viewed using epifluorescence microscopy with the oxidized probe fluorescing green.
Using the information from the
experiments, they determined that DAPG is more effective during early stages of
exponential growth as it interferes with mitochondrial function most likely via
depolarization of the mitochondrial membrane. However, the exact mechanism of
how DAPG interferes with mitochondrial function are still unknown, which could
be an area for future experiments to explore further. There is also the
interaction between plants and P.
fluorescens strains that exhibit DAPG production, where plants positively
favor and actually select for strains that produce DAPG, which means that there
are still the prospects of a plant-microbe interaction that could be looked
into, like low concentrations functioning as a signaling mechanism instead of
an antimicrobial.
Overall, this study is incredibly
promising as it not only helped identify a more specific mechanism behind the
prevention of further growth in fungi, but it also opened up the doors to more
specific areas of study. With the knowledge gained, we can now ask many more
questions about this curious secondary metabolite, like exactly how it interferes with a given microbe’s
mitochondria, and taking into account its dose-dependency and positive
selective pressure, whether or not it can actually function as a means to signal other microbes around it.
If
you would like to learn more about Pseudomonas
fluorescens, check out these cool links!
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