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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Impact of small molecule mediated cell-cell communication on the efficacy of inoculant bacteria in the rhizosphere

Background and objectives

Plant growth-promoting bacteria are used to enhance crop health and productivity. Currently, various nitrogen-fixing and biocontrol bacteria are employed or are being developed, to improve "environmentally friendly" agriculture. Clearly, the consequences of any such genetically modified bacteria must be researched with extreme care. It has recently been recognised that bacteria can function not only as individual cells but also as a multicellular group. This coordinated behaviour requires chemical secretions (of N-acyl homoserine lactones) by the bacteria into their environment. As the numbers of bacteria increase, these chemicals accumulate, activating the expression of bacterial genes that enable the population of bacteria to maximally exploit their environment. This process, known as “quorum-sensing” might, for example, enhance the bacteria's ability to form nitrogen fixing nodules on legumes or regulate production of antimicrobial agents that enable them to colonise roots and suppress pathogen growth. On the other hand, similar signals may be used by pathogenic bacteria to optimise their attack on plants. It is important to understand the potential impact of these communication systems in bacteria which may have agricultural purposes, both on other bacteria and on the environment.

This project aimed to provide a clearer understanding of the complex communications that exist in the rhizosphere (the environment surrounding the plant root). We examined the degree of potential communication (i.e. quorum-sensing cross-talk) between specific beneficial and detrimental bacteria. We also studied the role of quorum-sensing signalling in the production of antifungal and other metabolites that may limit plant pathogen growth, and thus the efficacy of biofertiliser and biocontrol bacteria.
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  Model rhizosphere system. image Model rhizosphere system.


Approach and methodology

Initially, detailed analysis of quorum-sensing based control was made in three bacterial species. We used one strain of the biofertiliser bacterium Rhizobium leguminosarum and two biocontrol bacteria, Pseudomonas chlororaphis PCL1391 and Pseudomonas fluorescens F117. Quorum-sensing based systems were identified in each bacterium, and R. leguminosarum was found to have four networked quorum-sensing systems. Bioassay test systems were established that allowed detection of quorum-sensing dependent cross-talk between different bacteria in vivo.
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  Nitrogen-fixing nodules on leguminous plant roots. image Nitrogen-fixing nodules on leguminous plant roots.
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  Bioassay of AHLs-separated by chromatography. image Bioassay of AHLs-separated by chromatography.


Main findings and outcome

Analysis of the effects of mutations in the R. leguminosarum quorum-sensing gene control systems revealed that legume nodulation, bacterial survival, horizontal gene transfer and the ability to inhibit growth of other rhizobia were all influenced by quorum-sensing. Studies on the two Pseudomonas species revealed that the production of antifungal antibiotics is controlled by quorum-sensing. Thus, in P. chlororaphis, quorum-sensing controls the production of phenazine-1-carboxamide, a compound responsible for the ability of this bacterium to inhibit growth of the root rot fungus Fusarium oxysporum. In P. fluorescens, quorum-sensing was shown to affect the regulation of the antifungal metabolite 2,4-diacetyl phloroglucinol.

Comparison of the quorum-sensing metabolites produced by these three bacteria and analysis of their regulatory effects, revealed that these growth promoting bacteria have the potential to cross-regulate via cross-talk in the rhizosphere. Significantly, they also have the potential to activate gene expression in plant pathogens such as Agrobacteria and Erwinia subspecies. Further work on a variety of soil isolates revealed that quorum-sensing systems are commonly found in diverse soil bacteria. In addition, some bacteria have the potential to use the quorum-sensing signalling molecules as a growth substrate.

In test systems, bioassays have been shown to be both sensitive and a good means of measuring communication between rhizosphere bacteria. Analysis of quorum-sensing based regulatory systems in artificial soil environments revealed that quorum-sensing signals produced by R. leguminosarum can positively affect gene transfer between soil bacteria of different genera. Direct effects of R. leguminosarum on plant pathogenicity by root pathogenic bacteria were not observed.
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Discovering quorum sensing in Pseudomonas
fluorescens
DF113

Panel A: TLC plate overlaid with Chromobacterium violaceum CV026.
 
   

Panel A: TLC plate overlaid with Chromobacterium violaceum CV026.
Lane 1, C6-HSL, Lane 2, Crude dichloromethane (DCM) extract of P. flurorescens culture supernatant; Lanes 3-7, HPLC fractions of the crude DCM extract. Note the appearance of spots corresponding to C6-HSL and C8-HSL in lanes 3 and 4.

   
Panel B: False pseudocolour image of an agar plate assay for long-chain N-acylhomoserine lactones (AHLs)    
     

Panel B: False pseudocolour image of an agar plate assay for long-chain N-acylhomoserine lactones (AHLs)
detected using a recombinant bioluminescent E. coli AHL sensor. Activity can clearly be seen in wells 6, 7 and 8.

     
Panel C: Cloning an AHL synthase from P. fluorescens.    
     

Panel C: Cloning an AHL synthase from P. fluorescens.
After introduction of a P. fluorescens gene library into a recombinant bioluminescent E. coli AHL biosensor, all colonies were patched on to agar plates and examined using a photon video camera for light output. A positive clone (top half) is compared with the AHL biosensor transformed with the plasmid vector alone (bottom half).

     
Panel D: Searching for the masking molecule(s).    
     

Panel D: Searching for the masking molecule(s).
False pseudocolour image of an agar plate overlaid with a short-chain recombinant E. coli AHL biosensor. The crude DCM extract was separated into 7 fractions by HPLC prior to re-mixing 6/7 fractions. In well 8, all 7 fractions were re-mixed, while a single fraction was omitted for each of the other wells. The greatest activity is observed when fractions 1 or 2 (wells 1 and 2 respectively) are omitted suggesting that these may contain the masking molecule(s).


Conclusions

This study revealed that several plant growth promoting bacteria can use quorum-sensing based signalling to communicate, thereby enhancing their efficacy in the rhizosphere. In particular, the production by Pseudomonas subspecies of antifungal metabolites which affect this signalling was noticeably affected. Furthermore, there is clear evidence that many soil bacteria use related quorum-sensing regulatory molecules and that there is the potential for chemical communication between bacteria of different species. This highlights the need to carefully consider the possible effects, notably with respect to safety issues, of introducing genetically modified bacteria for agricultural purposes.

 

Major publications

Andersen J.B., Sternberg C., Poulsen L.K., Bjørn S.P., Givskov M., Molin S., “New unstable variants of green fluorescent protein for studies of transient gene expression in Bacteria”.
Appl. Environ. Microbiol., 64, 1998, pp. 2240-2246.

Chin-A-Woeng T.F.C., Bloemberg G.V., van der Bij A.J., van der Drift K.M.G.F., Schripsema J., Kroon B., Scheffer R.J., Keel C., Bakker P.A.H.M., Tichy H.V., de Bruijn F.J., Thomas-Oates J.E., Lugtenberg B.J.J., “Biocontrol by phenazine-1-carboxamide-producing Pseudomonas chlororaphis PCL1391 of tomato root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici”.
Molec. Plant Microbe Interact, 11, 1998, pp. 1069-1077.

Delany I., Sheehan M.M., Fenton A., Bardin S., Aarons S., O’Gara F., “Regulation of production of the antifungal metabolite 2.4-diacetylphloroglucinol in Pseudomonas fluorescens F113: genetic analysis of phlF as a transcriptional repressor”.
Microbiology, 146, 2000, pp. 537-546.

Lithgow J.K., Wilkinson A., Hardman A., Rodelas B., Wisniewski-Dyé F., Williams P., Downie J.A., “The regulatory locus cinRI in Rhizobium leguminosarum controls a network of quorum sensing loci”.
Molec. Microbiol., 37, 2000, pp. 81-97.

Rodelas B., Lithgow J.K., Wisniewski-Dye F., Hardman A., Wilkinson A., Economou A., Williams P., Downie J.A., “Analysis of quorum-sensing-dependent control of rhizosphere-expressed (rhi) genes in Rhizobium leguminosarum bv. Viciae”.
Journal of Bacteriology, 181, 1999, pp. 3816-3823.
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imageResearch project
 

Contract number
BIO4-CT96-0181

Period
September 1996 -
December 1999

Coordinator
J.A. Downie
John Innes Centre
Norwich (UK)

 
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Partners


A. Hardman
University of Nottingham (UK)

B.J.J. Lugtenberg
Leiden University (NL)

F. O’Gara
University College Cork (IE)

M. Givskov
Technical University of Denmark
Lyngby (DK)

Y. Dessaux
CNRS, Institut des Sciences Végétales
Gif-sur-Yvette (FR)

A. Squartini
Università di Padova (IT)

B. Knight
Micro Bio Ltd
Hemel Hempstead (UK)

P. Williams
University of Nottingham (UK)

D. Grogan
Irish Sugar plc.
Carlow (IE)

 
 
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