Progress 12/01/01 to 11/30/04
Outputs
Reporter fusion studies provided evidence that synthesis of the symbiotic EPSII of Sinorhizobium meliloti is quorum sensing regulated in the bacterium via the receptor ExpR. Proteomic analysis revealed that over 100 proteins in the S. meliloti wildtype strain 1021 were significantly altered in accumulation by the addition of either C14-homoserine lactone (HSL) or 3-oxo-C16:1-HSL, or by the transition from early log phase to stationary phase, helping to identify functions that are subject to quorum sensing regulation in this symbiotic bacterium. Similarly, over 60 proteins in S. meliloti were differentially accumulated in a strain expressing AiiA, a lactonase from Bacillus that inactivates AHL signals, relative to the wildtype control with normal levels of N-acylhomoserine lactone (AHL) quorum sensing signals. The AHL-deficient strain was also altered in its symbiotic behavior and genetic switching. Cells of S. meliloti strain AK631 were found to produce a dozen
different AHLs, several of which were novel and only 4 of which were produced in common with strain 1021. The kinds and amounts of AHL quorum sensing signals produced by S. meliloti were quite dependent on culture medium and conditions. Medicago truncatula, the model legume host that is nodulated by S. meliloti, was found to produce over a dozen chromatographically separable compounds that mimicked the activity of bacterial AHL quorum sensing signals. These AHL mimic compounds were specific for interactions with particular bacterial AHL receptors and partitioned into organic solvents differently than bacterial AHLs, indicating that they are synthesized by the plant and capable of disrupting or modifying quorum sensing regulation in associated bacteria. The array of mimic compounds changed as seedlings developed and was different inside plant tissues compared to the secreted mimics. M. truncatula was also found to respond to physiological, nanomolar concentrations of bacterial AHLs in
a very extensive and specific manner. About 5% of the 2,000 root proteins resolved by 2D gel electrophoresis were changed over 4-fold in accumulation after exposure to AHLs. This is the first demonstration that eukaryotes can detect and respond globally to normal levels of bacterial quorum sensing signals. Plant responses to AHLs affected a wide diversity of functions, from defense and cytoskeleton to regulation, metabolism and the production of AHL mimic compounds. Quorum sensing-regulated proteins in S. meliloti were found to respond to a highly purified AHL mimic compounds from culture filtrates of the unicellular green alga Chlamydomonas reinhardtii. Two of the S. meliloti proteins affected by the algal mimic compound are essential for symbiotic N-fixation by the bacterium. The mimic compound was able to block the activating effects of the bacteriums own AHL signals on several quorum sensing regulation proteins.
Impacts
The studies conducted under sponsorship of this grant have identified: 1) more than a dozen different N-acyl homoserine lactone (AHL) quorum sensing signlas produced by two strains of the symbiotic N-fixing bacterium Sinorhizobium meliloti and 2) a broad diversity of functions under quorum sensing regulation via specific AHL signals and AHL receptors in S. meliloti. These studies help to establish which symbiotic behaviors in the bacterium are regulated or modulated by quorum sensing. The studies conducted under this grant also established that Medicago trunctula, the legume host of S. meliloti, produces a complex array of compounds that specifically mimic bacterial AHL quorum sensing signals. The production and secretion of such AHL mimic compounds potentially provides the host plant with the means to disrupt or manipulate quorum sensing regulation in the bacteria that it encounters. Such compounds could have broad medical applications in dealing with antibioitic
resistant bacterial pathogens and in breeding for healthier crops. This grant also supported studies that first demonstrated that eukaryotes can listen to the quorum sensing signal converstations of bacteria and respond extensively to the AHL signals it detects. This discovery may have many applications in using bacterial quorum sensing signals to trigger specific responses in eukaryotic hosts. It also reveals a new level of interaction between bacteria and their plant hosts.
Publications
- Rolfe, B.G., Mathesius U., Djordjevic, M., Weinman, .J, Hocart, C., Weiller G., and Bauer, W.D. 2003. Proteomic analysis of legume-microbe interactions. Comparative and Functional Genomics. 4: 225-228.
- Pellock, B.J., M. Teplitski, W.D. Bauer, R.P. Boinay and G.C. Walker. 2002. A LuxR homolog controls production of symbiotically active EPS II by Sinorhizobium meliloti. J. Bacteriol. 184:5067-5076.
- Bauer, W.D. and J.B. Robinson. 2002. Disruption of bacterial quorum sensing by other organisms. Current Opinon in Biotechnology 13:234-2
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Progress 12/01/01 to 11/30/03
Outputs
Ee showed previously that plants produce compounds that act like the N-acyl homoserine lactone (AHL) quorum sensing signals that many bacteria use to coordinate gene expression (Teplitski et al, 2000). The AHL signal-mimic compounds from the model legume Medicago truncatula and the unicellular green alga Chlamydomonas reinhardtii were systematically extracted and separated (7,9). The secretion of some mimic activities was found to require prior exposure to bacteria and the secretion of specific mimics changed markedly during seedling development. Proteomic analysis revealed that over 100 proteins in Sinorhizobium meliloti, the nitrogen-fixing symbiont of M. truncatula, were differentially accumulated in response to two of the bacterium's AHLs (4). The AHL receptor ExpR was found to mediate synthesis of exopolysaccaride in S. meliloti (1). M. truncatula responded to nanomolar levels of AHLs through the differential accumulation of over 150 proteins and the secretion of
different amounts or kinds of AHL signal-mimics (3). AHLs also induced new gene expression in a gene- and tissue-specific manner in white clover. The model nematode Caenorhabditis elegans also responded to nanomolar levels of AHLs in accumulation of over 30 proteins, and AHLs were potent chemoattractants. An AHL mimic from the unicellular green alga Chlamydomonas was shown to affect the accumulation of over 20 AHL-regulated proteins in S. meliloti (9). Additional AHL mimic activities from alfalfa seed were recovered and separated. Proteomic analysis revealed that over 60 proteins in S. meliloti, were differentially accumulated in a strain expressing AiiA, a lactonase from Bacillus that inactivates AHL signals. Cells of S. meliloti strain AK631 were found to produce a dozen different AHLs (6).
Impacts
Proteomic analysis revealed that over 60 proteins in S. meliloti were differentially accumulated in a strain expressing AiiA, a lactonase from Bacillus that inactivates AHL signals. Cells of S. meliloti strain AK631 were found to produce a dozen different AHLs.
Publications
- Mathesius, U., S. Mulders, M. Gao, M. Teplitski, G. Caetano-Anolles, B. G. Rolfe and W. D. Bauer. 2003. Extensive and specific responses of a eukaryote to bacterial quorum sensing signals. Proc. Nat. Acad. Sci. 100:1444-1449.
- Chen, H. Teplitski, M., M. Gao, J. B. Robinson, B. G. Rolfe, and W. D. Bauer. 2003. Proteomic analysis of wildtype Sinorhizobium meliloti responses to N-acyl homoserine lactone quorum sensing signals. J. Bacteriol. 185:5029-5036.
- Von Bodman, S.B., W.D. Bauer and D.L. Coplin. 2003. Quorum sensing in plant-pathogenic bacteria. Annu. Rev. Phytopathol. 41:12.1-12-28.
- Teplitski, M., M. Gronquist, A. Eberhard, M. Gao, J. B. Robinson, B. G. Rolfe, and W. D. Bauer. 2003. Chemical identification of N-acyl homoserine lactone quorum sensing signals produced by Sinorhizobium meliloti strains in defined medium. Arch. Microbiol. In press.
- Gao, M., M. Teplitski, J. B. Robinson and W. D. Bauer. 2003. Production of substances by Medicago truncatula that affect bacterial quorum sensing. Mol. Plant-Microbe Interact. 16:827-834.
- Hirsch, A.M., W.D. Bauer, D.M. Bird, J. Cullimore, B. Tyler and J. I. Yoder. 2003. Molecular signals and receptors controlling rhizosphere interactions between plants and other organisms. Ecology 84:858-868
- Teplitski, M., H. Chen, S. Rajamani, M. Gao, M. Merighi, R.T. Sayre, J.B. Robinson, B.G. Rolfe and W.D. Bauer. 2003. Chlamydomonas secretes compounds that mimic bacterial signals and interfere with quorum sensing regulation in bacteria. Plant Physiology In press.
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Progress 01/01/02 to 12/31/02
Outputs
We showed previously that plants produce compounds that act like the N-acyl homoserine lactone (AHL) quorum sensing signals that many bacteria use to coordinate gene expression. The AHL signal-mimics from the model legume Medicago truncatula were systematically extracted and separated. The secretion of some mimic activities required prior exposure to bacteria and the secretion of specific mimics changed markedly during seedling development. Proteomic analysis revealed that over 100 proteins in Sinorhizobium meliloti, the nitrogen-fixing symbiont of M. truncatula, were differentially accumulated in response to two of the bacterium's AHLs. M. truncatula responded to nanomolar levels of AHLs through the differential accumulation of over 150 proteins and the secretion of different amounts or kinds of AHL signal-mimics. AHLs also induced new gene expression in a gene- and tissue-specific manner in white clover. An AHL mimic from the unicellular green alga Chlamydomonas was
shown to affect the accumulation of over 20 AHL-regulated proteins in S. meliloti.
Impacts
The identification of extensive plant responses to bacterial AHL quorum sensing signals indicate that plant hosts have sophisticated and previously unknown mechanisms for detecting, counting and categorizing the bacteria they encounter. Since bacterial pathogens generally need quorum sensing signals to coordinate their attack on plant and animal hosts, host responses to these signals could be unavoidable and and thus a significant factor in establishing resistance.
Publications
- Pellock, B.J., Teplitski, M., Bauer, W.D., Boinay, R.P. and Walker, G.C.. 2002. A LuxR homolog controls production of symbiotically active EPS II by Sinorhizobium meliloti. J. Bacteriol. 184:5067-5076.
- Bauer, W.D. and Robinson, J.B. 2002. Disruption of bacterial quorum sensing by other organisms. Current Opinon in Biotechnology 13:234-237
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