What is the simplest, quickest, and/or cheapest way of finding out whether a molecule is involved in a novel quorum sensing / cell-cell signaling / signal transduction pathway?
Most literature explains assays that build on systems that have already been established, involving AHLs, AI-2, DKPs, DSFs, HAQs, etc., but our suspected signal molecule is not similar to those. It might be involved in cessation of cell growth, and we sort of have growth curves at different concentrations of the compound.
Outside of forward and reverse genetics, which would be especially difficult for our organism, an Archaeal methanogen, I’ve seen the most plausible options in this paper:
https://www.ncbi.nlm.nih.gov/pubmed/?term=Chemical+methods+to+interrogate+bacterial+quorum+sensing+pathways.
Affinity chromatography and photo-affinity labeling don’t seem simple or quick or cheap, though.
I’d appreciate any suggestions.
I think the problem is your question is much to open ended.
Lets assume your molecule is antibiotics (as such are screated by many microorganism) , e.g. in high enough concetration it kill the other cells. It is effecting the cell, and could cause difference in transcription if concentration is bellow MIC, but would you call it QS signal? And if it does indeed cause growth cessation why assume it is anything beside toxic waste/antibiotics?
I do not think there is a "simple" answer here. I suggest you start by reading the original papers describing the finding of the QS signal, to better understand the logic they used and start designing your experiments from there.
Yoram
Dear Elisa,
It is always difficult to go from growth experiments to the identification of an active compound, especially if your model organism is hard and slow to grow, you lack a fast and simple assay for the activity, and if you are not based in a biochemical lab. However, there are some simple experiments that you can do to narrow down the nature of your active compound.
I would suggest that you try some simple growth addition experiments to demonstrate that the effect is real, and that cells are responding to this compound in a dosage-dependent manner. The easiest experiment is to add some filter-sterilised (FS) culture supernatant from cultures in which you see cell death or growth-inhibition to a fresh culture - you would expect to be able to inhibit the growth of this or see cell death occurring earlier than if you did not add FS culture supernatant.
If this works, then there are some simple tests you can do : test FS culture supernatants from cultures grown in different media, grown for different lengths of time, and grown to different cell densities. Try fractionating your FS culture supernatant using dialysis membrane or filters (with different pore sizes) to see if you can put a size limit on the active compound; try heat inactivating the compound, and precipitating or extracting it with alcohol, phenol, ammonium sulphate, etc.
The aim of all of this is to produce a semi-purified concentrated extract that you could then consider fractionating by HPLC and then identifying the compound by mass spectroscopy (if you had access tho this type of analytical equipment). Being able to demonstrate that you have a semi-purified extract plus a simple phenotype you can assay activity with would be a good end-point for a project where you could not do any further biochemical analyses.
Regards, Andrew.
Hello,
Thank you both for your help. We’ve been able to isolate the compound, and identify it by mass spectroscopy, and are still working out what the causes and effects of it being there are.
Our basic growth curves at diferent concentrations of it do look similar to MIC graphs for antibiotics; we haven’t tried other growth tests yet - Thank you for the suggestion, Dr. Spiers. Searching literature, it so far seems that the only example growth cessation in quorum sensing involves rhizobia (with AHL / luxR homologue) trying not to overwhelm the immune system of their plant symbiont (http://jb.asm.org/content/178/2/372.full.pdf1, http://jb.asm.org/content/178/2/366.short), so it seems likely it has a different origen (maybe purpose), as Dr. Gerchman says.
(We’re holding out for it having a purpose, espcially since methanogens have so little room for waste, but we’ll see what we find.)
Thanks again.
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