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

Leland S. Pierson III.  Department of Plant Pathology, University of Arizona, Tucson, AZ 85721-0036.

Pierson, L.S., III. 2000. Bacterial Signaling: Identification of N-Acyl-Homoserine Lactone-Producing Bacteria. The Plant Health Instructor. DOI: 10.1094/PHI-I-2000-1214-01
Updated, 2005

OBJECTIVES:

To determine whether different bacterial populations have the ability to cross-communicate via diffusible signal molecules.

• Isolate bacteria from plant surfaces.
• Screen isolated bacteria for diffusible N-acyl-homoserine lactone (AHL) signal production using two isogenic derivatives of an AHL reporter strain.

INTRODUCTION:

For most of the modern era of microbiology and plant pathology, scientists have studied bacteria as pure cultures, arguing that the study of mixed populations was too complex and the results obtained undecipherable. This approach has resulted in detailed studies of isolated bacteria and the resultant idea that bacteria act as single cells, each cell sensing and responding to its environment independently. However, bacteria do not exist as isolated pure cultures in nature. Thus, studies that fail to consider the influence of the microbial community by default must be incomplete. Recently, studies have demonstrated that bacteria do not always act as single cells but have the ability to act as a population analogous to a multicellular organism².

In vitro studies have demonstrated that bacteria utilize a diverse range of signals, including their nutritional status and population density, to sense and respond to their biotic environment^4,11,12. These signals modulate the expression of a number of bacterial behaviors, including multicellular differentiation, fruiting body development, and sporulation, as well as processes such as bioluminescence, plasmid conjugal transfer, and secondary metabolism (Table 1).

Table 1. Examples of diffusible signals utilized by bacteria.

One well-characterized class of diffusible signal molecules are the N-acyl-homoserine lactone molecules (AHLs)^1,3,10 (Figure 1). These signals are used by a diverse range of gram-negative bacteria to regulate the expression of genes involved in both beneficial and detrimental plant-microbe and microbe-microbe interactions.

Click here for Figure 1.

MODEL FOR AHL GENE REGULATION

AHL-mediated gene regulation is also known as cell density-mediated gene regulation and quorum sensing. There are several excellent reviews on AHL-mediated gene regulation^{1, 2, 3}. A simplified model for AHL-mediated gene regulation is given in (Figure 2). In this general model, two genes are important. The first, the R gene, encodes a transcriptional regulatory protein that is required to activate the target gene(s) but is unable to do so by itself. The second gene, the I gene, encodes an enzyme (AHL synthase) that synthesizes a small diffusible AHL signal from the cellular precursors S-adenosyl-methionine (AdoMet) and a fatty acyl ACP^5,7. The AHL signal can freely diffuse out of and into the bacterial cell. The key point is the R regulatory protein is active only when it is binds the AHL signal. At low cell densities, the AHL signal diffuses out of the cell, thus reducing its intracellular concentration below that sufficient to activate the R regulatory protein. When bacteria are in a confined space or at high cell density, the AHL intracellular concentration threshold is exceeded and the now activated R regulatory protein stimulates (or represses) transcription of the target genes.

Figure 2 Click image for an enlarged view and more information.

It is important to realize that each R regulatory protein recognizes only a subset of possible AHL signals. Thus, failure to activate an AHL reporter strain is not evidence of a lack of AHL production by the test strain, only that the R regulatory protein of the AHL reporter strain failed to recognize the signal produced by the test strain.

AN AHL REPORTER STRAIN

AHL regulatory systems have been demonstrated in a number of plant-associated bacteria ^3,10,13 including Pseudomonas aureofaciens strain 30-84. This root-colonizing strain is an effective biological control agent for take-all disease of wheat caused by the ascomycete Gaeumannomyces graminis var. tritici (Ggt). Disease suppression results primarily from the production of phenazine antibiotics⁹. In addition to their role in disease suppression, phenazines enhance the survival of P. aureofaciens in the rhizosphere in competition with indigenous microorganisms⁶. The expression of the phenazine biosynthetic operon (phzXYFABCD) is controlled by PhzI¹⁴ and PhzR⁸. What makes this a useful reporter for the presence of AHL signals is that the phenazines are bright orange compounds easily visible without special chromogenic indicators. Wood et al.¹³ demonstrated that AHL signals produced by an isogenic donor population restored phenazine gene expression in a phzI mutant population on wheat and confirmed that AHL signals are shared between populations in the rhizosphere. Subsequent work, in which a rhizobacterial library generated from wheat plants was used, demonstrated that approximately 8% of the rhizosphere bacteria of wheat also positively activated phenazine gene expression via the production of AHL signals¹⁰.

In this laboratory exercise, you will screen plant-associated bacteria for their ability to produce AHL signals recognized by the AHL reporter strain P. aureofaciens 30-84I. The strain is a derivative of P. aureofaciens 30-84 in which the phzI gene (encoding the endogenous AHL synthase) has been inactivated¹⁴. Since the accumulation of AHL signal is a requirement for phenazine gene expression, strain 30-84I is unable to produce its own AHL signal and appears white due to the lack of production of the orange phenazines. However, some of the exogenous AHL signals produced by other bacteria can restore the ability of strain 30-84I to produce phenazines, resulting in the production of an orange halo.

In addition, some AHL signals produced by other bacteria could serve to block normal phenazine activation by strain 30-84. To examine this possibility, you will also test the effect of the other bacteria on strain 30-84 and look for the inhibition of phenazine production.

MATERIALS (per group)

• Selection of recently collected plant roots and leaves. These should be stored in plastic bags at 4ºC until use.
• Vortex mixer
• Sonicating water bath (optional)
• 10-ml tube of sterile phosphate buffered saline (PBS)
• Sterile 1.5-ml snap-top microfuge tubes (have containers full of open sterile microfuge tubes)
• Pipetor (P-20 & P-100) and a box of sterile yellow tips
• 6 LB + Cycloheximide (100 mg/ml) agar plates (Cycloheximide is an antibiotic that prevents the growth of fungi)
• 3 LB agar plates
• 20-ml LB broth (bottles)
• Jar of ethanol plus glass spreader

CLICK HERE FOR MEDIA RECIPES (buffer, LB media), PROTOCOL (glass spreaders), and BACTERIAL STRAINS

EXPERIMENTAL PROTOCOL

Isolate and plate bacteria from plant surfaces
(Figure 3)

Figure 3 Click image for enlarged view and more information

• Collect several samples of different plants, both roots and leaves. Store in labeled plastic bags at 4ºC.
• Take about 1 g each of your different plant materials, and cut both roots and leaves separately into small pieces with a sterile razor blade. Place the pieces of each sample into separate, sterile glass test tubes. Add 2 ml of sterile PBS buffer to each test tube.
• Vortex each tube for 5 sec, sonicate 30 sec. Repeat (the sonication step can be omitted but some of the more tightly adhering bacteria may not be recovered).
• Serially dilute each to 10^-2 in sterile microfuge tubes (each 1/10 dilution is achieved by adding 100-µl sample to 900-µl sterile PBS) and plate on LB + Cycloheximide plates at 10^-1, 10^-2, and 10^-3.
• Incubate at 28ºC up to 3 days.

Next Period: Inoculating cultures for AHL screen plates (Figure 4)

Figure 4 Click image for enlarged view and more information

• After sufficient numbers of colonies have appeared, pick 12 (or more) isolated colonies that appear distinctly different in shape, color, etc. (this is your test strain collection). At the same time, transfer by picking a colony and touching it onto an LB agar plate and then using the same toothpick to inoculate 1-ml LB cultures of each test strain in sterile glass test tubes. Shake at 28ºC overnight.

• Inoculate 1-ml LB cultures of strains 30-84, 30-84Ice, 30-84Gac, & 30-84I. Shake at 28ºC overnight. Strains 30-84 and 30-84I are the AHL reporter strains, while strains 30-84Gac and 30-84Ice are control strains.

Next Period: Preparation of AHL screen plates

Figure 5. Arrangement of spot tests on LB agar plates seeded with either strain 30-84 or strain 30-84I. There should be two LB agar plates, one seeded with strain 30-84 and a second plate seeded with strain 30-84I.

• Use overnight cultures of strains 30-84 and 30-84I only for this step (Figure 5).

1. Aseptically transfer 1 ml of strains 30-84 and 30-84I to separate sterile 1.5-ml microfuge tubes.
2. Microfuge (max. speed) 1 min. Aseptically remove each supernatant.
3. Resuspend each pellet in 500 µl of LB broth using a pipetor with a sterile tip.

• Repeat steps 2 and 3.
• Make an orientation mark on the outside of each plate as shown in Fig. 5.
• Using an ethanol-flamed glass spreader, uniformly spread 100 µ of each culture from step 4 above onto a separate LB agar plate. Let the remaining liquid soak into the plate. These plates are now seeded with a lawn of strain 30-84 or strain 30-84I.

• Spot 5 µl of the overnight culture of each test isolate onto the lawn. Include strain 30-84Ice as a positive control (produces an AHL signal recognized by strain 30-84I) and strain 30-84Gac as a negative control (does not produce any AHL signal). Let spots soak into the plate. Store the remaining overnight cultures at 4ºC for possible later use.

• Incubate plates at 28ºC.

Next Periods (2-3 days): Scoring screening plates for cross-communication

Figure 6 Click image for enlarged view and more information

• Score the plate seeded with strain 30-84I for the presence of an orange halo surrounding the test spots. The presence of orange halos surrounding the test spots indicates restoration of phenazine production by a signal produced by the test spots (positive cross-communication) (Figure 6A).

• Score the plate seeded with strain 30-84 for the presence of a white halo surrounding the test spots. The presence of white halos surrounding the test spots indicates inhibition of phenazine production by strain 30-84 by a signal produced by the test spots (negative cross-communication) (Figure 6B).

• Streak out the positively or negatively cross-communicating strains from the LB agar plate you made in Figure 4.

DATA COLLECTION

1. Set up a score sheet similar to the one illustrated below.
2. Note the plant source (name if known) and number the isolates for each.

For example, name isolates P1, P2, P3, etc. Score the effect of each test strain on 30-84I or 30-84 as follows below.

QUESTIONS FOR THOUGHT

1. What is the percentage of isolates from each plant part (leaves or roots) capable of cross-communicating with 30-84I? with 30-84?

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

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

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

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?

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

Instructors can find suggested answers to these discussion questions here. 

LITERATURE CITED

1. Dunlap, P.V. 1997. N-acyl-L-homoserine lactone autoinducers in bacteria: Unity and diversity. Pages 69-106 in: Bacteria as Multicellular Organisms. J.A. Shapiro and M. Dworkin, eds. New York University Press, New York.
2. Dunny, G.M., and  S.C. Winans.  1999. Bacterial life: Neither lonely nor boring. Pages 1-5 in: Cell-Cell Signaling in Bacteria. G.M. Dunny and S.C. Winans, eds. American Society for Microbiology Press, Washington, D.C.
3. Fuqua, C., S.C. Winans, and E.P. Greenberg. 1996. Census and consensus in bacterial ecosystems: The LuxR-LuxI family of quorum sensing transcriptional regulators. Annu. Rev. Microbiol. 50:727-751.
4. Gray, K.M. 1997. Intercellular communication and group behavior in bacteria. Trends Microbiol. 5:184-188.
5. Hanzelka, B.L., and E.P. Greenberg. 1996. Generation of cell-to-cell signals in quorum sensing: Acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein. Proc. Natl. Acad. Sci. 93:9505-9509.
6. Mazzola, M., R.J. Cook, L.S. Thomashow, D.M. Weller, and L.S. Pierson III. 1992. Contribution of phenazine antibiotic biosynthesis to the ecological competence of fluorescent pseudomonads in soil habitats. Appl. Environ. Microbiol. 58:2616-2624.
7. Moré, M.I., L.D. Finger, J.L. Stryker, C. Fuqua, A. Eberhard, and S.C. Winans. 1996. Enzymatic synthesis of a quorum-sensing autoinducer through use of defined substrates. Science 272:1655-1658.
8. Pierson, L.S., III, V. D. Keppenne, and D.W. Wood. 1994. Phenazine antibiotic biosynthesis in Pseudomonas aureofaciens 30-84 is regulated by PhzR in response to cell density. J. Bacteriol. 176:3966-3974.
9. Pierson, L.S., III, and L.S. Thomashow. 1992. Cloning and heterologous expression of the phenazine biosynthetic locus form Pseudomonas aureofaciens 30-84. Mol. Plant-Microbe Interact. 5:330-339.
10. Pierson, L.S. III, D.W. Wood, E.A. Pierson, and S.T. Chancey,  1998. N-acyl-homoserine lactone-mediated gene regulation in biological control by fluorescent pseudomonads: Current knowledge and future work. Eur. J. Plant Pathol. 104:1-9.
11. Schell, M.A. 2000. Control of virulence and pathogenicity genes of Ralstonia solanacearum by an elaborate sensory array. Annu. Rev. Phytopathol. 38:263-292.
12. Wirth, R. 2000. Sex pheromones and gene transfer in Enterococcus faecalis. Res. Microbiol. 151:493-496.
13. Wood, D.W., F. Gong, M.M. Daykin, P. Williams, and L.S. Pierson III. 1997. N-acyl-homoserine lactone-mediated regulation of phenazine gene expression by Pseudomonas aureofaciens 30-84 in the wheat rhizosphere. J. Bacteriol. 179:7663-7670.
14. Wood, D.W. and L.S. Pierson III. 1996. The phzI gene of Pseudomonas aureofaciens 30-84 is responsible for the production of a diffusible signal required for phenazine antibiotic production. Gene 168:49-53.

ACKNOWLEDGEMENTS
Some of this work was supported by grant No. 98-35303-6403 from the National Research Initiative Competitive Grants Program of the U.S. Department of Agriculture.