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Bacterial Signaling: Identification of N-Acyl-Homoserine Lactone-Producing Bacteria

by Leland S. Pierson III.

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Questions for Thought

1. What is the percentage of the selected isolates from each plant was capable of cross-communicating with 30-84I? with 30-84? (remember: we only sampled a very small number of possible isolates).

From Figure 7 we see two possible positive cross-communicators (7B) in addition to the positive control spot 30-84ICE. We also see one possible negative cross-communicator (7C) on the top right plate of 30-84.

2. A. What would happen if you picked strain 30-84I from the orange halo sector of the plate and re-streaked it onto LB agar by itself? B. Pick strain 30-84 from the white halo sector and streaked it by itself?

A. Strain 30-84I that turned orange is expected to occur because the test spot produced an AHL that is recognized by PhzR in 30-84I. One would predict that if 30-84I was streaked away from the test spot that it would not continue producing phenazines because the source of the AHL signal was removed.

B. Where strain 30-84 appears white indicates that a) it can still grow in the presence of the test spot (so its growth is not being limited), and b) that something produced by the test spot is blocking QS (e.g. QSI). One would predict if the white area of 30-84 were re-streaked apart from the test spot the QSI would cease and 30-84 would become orange again.

3. Why would different bacteria cross-communicate? That is, list several possible ecological function(s) of cross-communication.

It may be that each member of a microbial community contributes different traits that benefit the community. However, those traits may not need to be expressed constitutively, or only some need to be expressed, and certain levels, when needed. The ability to cross-communicate among members of a community enhances the complexities of the interactions and this may enhance the overall fitness of the community.

4. Discuss two experiments that you might perform to study the phenomenon of cross-communication further.

One could grow a positively- or a negatively-cross-communicating test strain separately, centrifuge and remove the bacterial cells, and see if the remaining supernatant had the same ability to activate or inhibit orange production in strain 30-84I or 30-84. This would demonstrate that the signal was present in the culture supernatants.

One could then quantify the effect of the cross-communication on the total amount of orange phenazine produced by extracting the pigment and quantifying using spectroscopy.

5. Does the fact that a test isolate produces an AHL signal in vitro prove that the same isolate produces an AHL signal in planta?

No, it only demonstrates that under the growth conditions used (LB broth in this case) that the test strain produced a signal recognized by the reporter. It is highly conceivable that several more of the test strains might produce AHL signals when grown in situ on the roots where the environment may favor signal production. This would be an exciting follow up set of experiments to perform.

6. Some of the test isolates might produce a clear zone in the lawn of P. chlororaphis. What might this clearing indicate about the interactions between the two strains?

The most likely explanation is that the test isolate is itself producing a compound that inhibits growth of the P. chlororaphis strain.

Also notice that some of the test spots failed to grow on the lawns of 30-84I and 30-84. Perhaps the growth of these test strains was being inhibited by P. chlororaphis in these cases.