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Monitoring Bacterial Cell-to-Cell Communication “Quorum sensing” Using FLUOstar OPTIMA

K.E. Eboigbodin, G.K. Robinson, Dept. of Biosciences, University of Kent, Canterbury, CT2 7NJ, 08/2009

  • Quorum sensing is a phenomenon allowing bacteria to adapt to environmental changes
  • Effects on quorum sensing were investigated by measuring bioluminescence and bacterial growth
  • The FLUOstar OPTIMA was used to measure absorbance and luminescence in parallel

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Introduction

Bacteria can monitor and respond to changes to environmental conditions via a cell density process known as quorum sensing (QS). Bacteria uses this process to monitor their community by producing, detecting and responding to low molecular mass signal molecules, called autoinducers (AI).(1) When the cell density increases the concentration of these signaling molecules will also increase. Once the accumulation of these molecules reaches a threshold, bacteria are collectively able to regulate gene expression and therefore cooperatively regulate their metabolic behavior. Quorum sensing has been shown to regulate a variety of processes in bacteria. These includes bioluminescence and symbiosis in Vibrio fischeri (2), expression of virulence genes in Pseudomonas aeruginosa (1), expression of virulence, surface proteins and biofilm formation in Escherichia coli (3), and biosynthesis of extracellular polymeric substances and pathogenicity in Erwinia stewartii (4). Two major types of quorum sensing molecules (QSMs) have been widely described in literature. The commonest QSM used by Gram-negative bacteria are known as N-acyl homoserine lactones (HSLs) whilst Gram-positive bacteria use amino acids and short peptides (oligo-peptide) as their AIs.

Quorum sensing was first described in V. fischeri, a Gram-negative bacteria. At low cell density, Vibrio fischeri is non-bioluminescent, but when the concentration increases (high cell density), the organism is bioluminescent. The molecular basis for regulation of bioluminescent in V. fischeri via quorum sensing has been well studied(5). The gene cluster responsible for light production consists of eight genes (luxA-E, luxG, luxI, and luxR) (Figure 2)(5). The regulator proteins responsible for quorum sensing in this organism are proteins encoded by luxI and luxR. LuxI encodes the enzyme AHL synthase, which catalyses the reaction involved in the biosynthesis of HSLs known as N-3-oxo-hexanoyl-L-homoserine lactone (3-oxo-C6-HSL). LuxR encodes the protein which binds to the AI and also activates the luxA-E and luxI operons.

Fig. 1: BMG LABTECH’s multidetection microplate reader FLUOstar OPTIMA

 


Fig. 2: The HSL-dependent LuxI/LuxR regulatory system of V. fischeri.

The aim of this study was to elucidate the effect of changes in environmental condition such as growth media as well as the addition of exogenous homoserine lactone on 3-oxo-C6-HSL on the growth and bioluminescence in V. fischeri. A FLUOstar OPTIMA (Fig. 1) was used to measure absorbance and luminescence in script mode.

Materials and Methods

Bacteria strain and Media

V. fischeri ESR1 and its mutants were kindly supplied by Prof Edward G. Ruby(6). The ESR1 variants include a signal-negative mutant that does not synthesize the 3-oxo-C6-HSL signal but can still respond to exogenous 3-oxo-C6-HSL, (V. fischeri KV240) and a signal-blind strain that produces 3-oxo-C6-HSL but do not respond to 3-oxo-C6-HSL (V. fischeri KV267).
All V. fischeri strains were grown in Luria-Bertani salt media (LBS), which contains 1% (wt/vol) tryptone, 0.5% (wt/vol) yeast extract (wt/vol), 2% NaCl (wt/vol), and 0.3% (vol/vol) glycerol in 50 mM Tris-HCl (pH 7.5) or in seawater tryptone (SWT) which contains 0.5% (wt/vol) tryptone, 0.3% (wt/vol) yeast extract, and 0.3% glycerol (vol/vol) in 70% seawater.

Evaluation of V. fischeri growth and bioluminescence under different media and concentrations of homoserine lactone
The growth and bioluminescence of all V. fischeri strains were monitored using a multi-mode plate reader (FLUOstar OPTIMA, BMG LABTECH). Briefly, overnight cultures of V. fischeri strainsgrown in the above media at 28°C were inoculated to fresh media (1:500 dilution) with or without the addition of exogenous 3-oxo-C6-HSL. 200 µL of diluted cells (quadruplicate) were then transferred into a white 96 well clear bottom (Greiner Bio-One, UK). The instrument incubation temperature was set at 28°C with continuous shaking at 300 rpm. The OD600 and luminescence was then measured every hour for at least 16 h. The V. fischeri bioluminescence reading was reported as relative light units (RLU) divided by OD600.

Results and Discussion

The increase in cell number over time for V. fischeri strains, grown in LBS and SWT can be seen in figure 3. The growth curves represent a typical growth curve under batch conditions with two clearly distinct phases i.e. exponential 2-8 h and onset of stationary phase after 8 h. There was no significant difference between each strain, suggesting that quorum sensing in V. fischeri does not promote or inhibit growth.


Fig. 3: Batch growth curve of V. fischeristrains grown in LBS and SWT with and without addition of 10 µM 3-oxo-C6-HSL.

The expression of bioluminescence by V. fischeri ESR1 cultivated in LBS and SWT (with or without 3-oxo-C6-HSL) can be seen in figure 4 and 5 respectively. For V. fischeri ESR1, bioluminescence was higher in SWT than in LBS. Earlier activation of bioluminescence was observed when exogenous 3-oxo-C6-HSL was added to V. fischeri ESR1 at the onset of growth. For V. fischeri KV240, bioluminescence was only observed when exogenous 3-oxo-C6-HSL was added at the onset of growth. This confirms that the strain lacks the ability to produce endogenous 3-oxo-C6-HSL and can only express bioluminescence upon addition of exogenous 3-oxo-C6-HSL. As expected V. fischeri KV267, did express bioluminescence with or without the addition of exogenous 3-oxo-C6-HSL since it lacks the ability to respond to the signal molecule.


Fig. 4: Bioluminescence expression of Vibrio fischeri strains grown in LBS
(+ means addition of 10 µM 3-oxo-C6-HSL; - means no 3-oxo-C6-HSL)


Fig. 5: Bioluminescence expression of V. fischeri strains grown in SWT
(+ means addition of 10 µM 3-oxo-C6-HSL; - means no 3-oxo-C6-HSL)

Conclusion

In this study, we show that the FLUOstar OPTIMA microplate reader is a useful tool for understanding quorum sensing in bacteria. The instrument is able to monitor microbial growth and bioluminescence in parallel.

The assay described in this application note would also greatly benefit from the Atmospheric Control Unit (ACU) and the ability to precisely control O2 and CO2 concentrations within the microplate reader.

References

  1. Waters, C. M., and Bassler, B.L. (2005) Quorum sensing: Cell-to-Cell Communication in Bacteria. Annu. Rev. Cell Dev. Biol. 21, 319-346.
  2. Miller, M. B. and Bassler, B.L. (2001) Quorum sensing in bacteria. Annu. Rev. Microbiol. 55, 165-199.
  3. Eboigbodin, K. E., Newton, J., Routh, A. and Biggs, C. (2006) Bacterial quorum sensing and cell surface electrokinetic properties. Appl. Microbiol. Biotechnol. 73, 669-675.
  4. Beck von Bodman, S., and Farrand, S.K. (1995) Capsular polysaccharide biosynthesis and pathogenicity in Erwinia stewartii require induction by an N-acylhomoserine lactone autoinducer. J. Bacteriol. 177, 5000-5008.
  5. Whitehead, N. A., Barnard, A.M. Slater, H. Simpson, N.J. and Salmond, G.P. (2001) Quorum-sensing in Gram-negative bacteria. FEMS Microbiol. Rev. 25, 365-404.
  6. Visick, K. L., Foster, J., Doino, J., McFall-Ngai, M. and Ruby, E.G. (2000) Vibrio fischeri lux genes play an important role in colonization and development of the host light organ. J. Bacteriol. 182, 4578-4586.