Visualization of the distribution pattern of signal-producing and degrading

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In nature, for the acyl-HSL signals to be appropriately functional as accurate reflection of the quorum sensing bacterial population, there must exist an elaborate balance between the bacteria that synthesize the signals and their possible acyl-HSL- degrading neighbors. It would be intriguing to study the crosstalk between acyl-HSL synthesizers and degraders. To initiate the investigations into this topic, it would be interesting to study the distribution pattern of signal-producing and -degrading bacteria.

Fluorescence in situ hybridization (FISH) is widely used to visualize the distribution of specific species in microbial ecosystems (14). The 16S rRNA-directed oligonucleotide DNA probes, which are specific to certain bacteria, e.g., Variovorax strain SOD31, and labeled at the 5’ with proper fluorophores such as fluorescein or 6- carboxyfluorescein (FAM), can be used to detect the corresponding bacteria. After fixation and permeabilization, FISH labeled bacteria can be visualized by confocal laser scanning microscopy. Colocalization of signal-producing and -degrading bacterial may demonstrate their relative distribution pattern.

Taking advantage of the Variovorax strain SOD31 that has remarkably high affinity to degrade acyl-HSL and possibly maintains a population in the turf soil, a soil sample can be carefully laid out on a glass slide and Variovorax strain SOD31 may be FISH labeled. Certain pattern of SOD31 distribution might be revealed. The idea is to see whether signal-degrading bacteria can cluster around signal-producing bacteria.

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Alternatively, by using a similar system to the biofilm reactors in Chapter 4, a glass microchamber can be applied instead of the columns that can be disconnected from the system for confocal microscopy visualization without disturbing the structure of the biofilm formed. Pantoea stewartii, which is a plant pathogen, produces 3-oxo-hexanoyl- HSL for quorum sensing, and is not known to degrade its own signal (15). We have observed that the bacterium may accumulate the signal to ca. 5 µM in laboratory cultures

in rich medium. The bacterium can be grown in the chamber supplied with relatively rich medium, e.g., yeast extract medium, for acyl-HSL to be accumulated in the chamber. Then culture of the signal-degrading bacterium Variovorax strain SOD31 can be added to the chamber for a period of time for them to utilize acyl-HSL. Then FISH will be performed on the bacterium in the chamber. The distribution pattern of these two bacteria may be observed under confocal microscope. Such may provide us information upon interspecies interactions.

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