The overall goal of the following experiment is to identify interactions between different microbes that are mediated by secreted compounds and lead to changes in bacterial gene expression. This is achieved by culturing of bacterial strain containing a fluorescent transcriptional reporter with environmental microbes as colonies on agar Petri plates. The orange circles represent colonies of the environmental microbes.
While the blue circles are colonies of the reporter, growth on solid media allows the metabolites that these colonies produce to diffuse through the agar and activate gene expression in nearby reporter colonies. The potential hits indicated here with stars are those colonies that may be secreting compounds that alter gene expression in the reporter. These organisms are isolated from the co-culture screen plates.
These potential inducers are retested in a secondary screen to confirm that they were the organisms secreting molecules that alter bacterial gene expression in the reporter. Although this method provides insight into the biofilm forming interactions of bacillus subtles with soil organisms, it can also be applied to other systems such as microbial interactions that affect virulence expression in staphylococcus or pseudomona species. Indeed, it can theoretically be applied to identify microbial interactions that alter gene expression for any reporter in any genetically tractable microbe After generating fluorescent reporter constructs for your organism of choice, according to the text protocol, collect soil by removing the top 0.5 centimeters of exposed surface soil before using a spatula to collect soil and conical tubes, add 0.85%sterile saline at a ratio of 10 milliliters per one gram of soil to make a soil slurry and vortex it for one minute to dislodge the bacteria from the soil particles.
After letting the soil slurry settle for one minute, transfer the upper aqueous layer to a fresh tube, dilute the reporter and soil samples according to the text protocol and spot 50 microliters of each onto the center of co-culture screen plates under conditions that will not activate the reporter construct alone. Also plate a soil sample alone and a reporter alone as controls. Add about 23 millimeter sterile glass beads onto each plate and spread the cells by keeping the plates on the bench top and shaking them back and forth.
While rotating until the liquid is absorbed, flip the plates over and discard the beads into a waste beaker containing ethanol. After incubating the co-culture plates using fluorescence and beginning from the top of the plate, slowly move the plate back and forth across the field of view to bright spots. After one full sweep, turn the plate 90 degrees and repeat the process.
When a fluorescent spot is detected slowly turn on bright field illumination and determine if the fluorescence is associated with a bacterial colony. To isolate putative inducing organisms, which will be the non fluorescent colonies proximal to the fluorescent colonies. Position them in the center of the field of view and accessible by your dominant hand, leaving the fluorescent lamp on.
Slowly turn on Brightfield to identify by shape and position the fluorescent colony and surrounding inducing organisms Using a glass rod, pick up a sterile round 200 microliter gel loading tip and hold it like a pencil. Rest your outer palm against the stage to stabilize it on the work surface. Then place your other hand on the inner thumb side of your hand to stabilize the picking tool, keeping the pipette tip above the plate surface.
Move it into the field of view and center it above the colony you would like to pick. The tip will be outta focus. Using the outer edge of your hand slowly pivot the pipette tip down to the colony to be picked.
Touch it very lightly trying to minimize how many other colonies the tip contacts without rotating the picking tool. Streak the tip onto a section of a fresh plate. After picking colonies, incubate plates according to the assay guidelines.
When colonies are visible, use a stereoscope to determine if single or multiple types of organisms are growing on the plate re streak Each morpho type onto a fresh plate and incubate until grown. To test whether putative inducers are true inducers, prepare lawns of the reporter and non fluorescent parental strains. After the plates dry, use sterile toothpicks to pick cells from test colonies.
Patch onto a blank plate and then onto the two lawn plates. Alternatively, resus, suspend the putative inducing organisms in one milliliter of liquid medium in a sterile plastic vet, and measure the OD 600 of the suspensions after normalizing the OD of each suspension to arrive at an OD 600 of 0.5 spot, one microliter of each suspension onto each of the three plates. Finally, after allowing the organisms to grow, use fluorescence under a dissecting microscope to identify putative inducing organisms that activate the fluorescent reporter strain, but not the parental control strain.
The screen here was used to identify soil organisms secreting compounds that alter gene expression of a structural component of the biofilm matrix in b subtles using a fluorescent transcriptional reporter. Ideally, each co-culture plate should contain a one-to-one ratio of reporter to soil colonies that are closely spaced individual colonies. Here we illustrate how having a high ratio of reporter colonies shown as blue circles relative to soil colonies shown as brown circles increases confidence in pinpointing the actual inducing organism from co-culture plates.
The inducing soil organisms secrete compounds that activate the reporter. Multiple reporter colonies will be induced on the plate with the higher reporter to soil ratio facilitating the identification of the true inducers shown in red from other putative inducers indicated by stars. The nutrient content controls the extent of growth colony formation, while the dilution of the inoculum determines whether the resulting colonies are appropriately dispersed.
As seen in this co-culture, image B subtles produces a fluorescent biofilm matrix in response to numerous microbes from the soil. For the soils we examined, we had a high hit rate for the Ptap A YFP matrix reporter. This is in contrast to our unpublished results from analogous screens.
Using the sporulation and competence reporters were few to no hits were identified. Thus, the HI rates for different cell types are highly variable and may be difficult to predict in advance. Shown here are two examples of co-culture screen plates before and after potential inducing colonies have been picked for isolation.
Negative and positive results from both the patch and spot method are shown here with approximately 50%of the colonies examined in our secondary screen being true positives. Following this procedure, other methods can be performed to answer additional questions, for instance 16 s SR DNA sequencing to determine the phylogenetic identity of the inducing organism, or using HPLC mass spectrometry in NMR to determine the chemical identity of the active compound.
