Progress 01/15/10 to 01/14/14
Outputs
Target Audience: Our target audience is our peer scientists in academia and industry who have an interest in cell to cell communication by bacteria as well as in Bradyrhizobium japonicum soybean symbiosis. We reached this target audience through publications, presentations at scientific meetings and face to face meetings with scientistis from Novozymes Corp. Changes/Problems: In the no-cost extension period we initiated work to define, at the genomic level, those B. japonicum genes whose expression and function are required to survive in seed and in soil, to compete with other rhizosphere bacteria, and to establish symbiosis with plant hosts. To do this we planned to apply an untargeted mutant analysis method called “Tnseq”. This method is currently used in Dr. Harwood’s lab and tracks the behavior of individual mutants in pools of strains carrying transposon insertions in nearly all nonessential genes. Using high-throughput sequencing techniques, we can measure the abundance of each mutant in populations that have been subjected to a variety of conditions (see specifics below). This approach will allow us to identify genes that are important to each condition because mutants defective in these genes will not survive as well as the wild-type. It turned out to be exceptionally difficult to generate a saturating transposon mutant library in B. japonicum. However we eventually prevailed. Our plan is to use descretionary funds, as they become available, to test the transposon library (approximately 100,000 random insertions in genome) for i) survival on soybean seeds, alone and in competition with other bacteria; ii) survival in soil, alone and in competition with other bacteria; and iii) in plant nodulation. A corollary benefit of the Tn-seq method is that it will also provide an inventory of B. japonocum essential genes, i.e. genes for which no Tn insertion is found under any condition. We know survival and competition in seeds and in soil are important, yet poorly understood, stages in the B.japonicum life cycle. Using an untargeted, high-throughput mutant analysis should provide novel candidate genes required for these lifestages. What opportunities for training and professional development has the project provided? Andrea Lindemann, Ph.D., post-doctoral fellow Nathan Ahlgren, Ph.D., post-doctoral fellow Daniel Mueller, visiting diploma student from Germany All three of these individuals were scientfically trained though this project. Dr. Lindemann isnow employed in the Biotech industry. Daniel Mueller entered a PhD program at the ETH in Switzerland. Dr. Ahlgren has taken a second postdoctoral fellowship in Biological Oceanography. How have the results been disseminated to communities of interest? We have disseminated our results through publications and presentations at meetings. We also initiated collaborations with Dr. Woo-Suk Chang (U. Texas, Arlington), Dr. Yaowei Kang (Novozymes), and Dr. Juan Quelas (Universidad Nacional de La Plata) to investigate the role of IV-HSL in plant infection, dessication resistance, and motility/biofilm formation. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Goal 1: We completed and published work describing our discovery of an unusual branched chain acyl-HSL QS signal from B. japonicum USDA110, isovaleryl-homoserine lactone (IV-HSL). This novel signal has several interesting features including the fact that its side-chain substrate is derived from a CoA-intermediate from branched amino acid biosynthesis, as well as the observation that it is synthesized and detected at concentrations much lower (~100-fold) than most acyl-HSL QS systems. Interestingly, B. japonicum can also detect straight chain acyl-HSLs made by other bacteria at higher (but still physiologically relevant) concentrations. Thus it can evesdrop on other bacteria. Goal 2. The identification of the B. japonicum QS regulon. This work required that we develop protocols for transcriptomic analyses by high-throughput sequencing (RNAseq) in B. japonicum. We designed a set of primers for RNAseq experiments that work well in B. japonicum. This method will be useful for many research groups beyond our own. Using our protocol, we identified 30 genes that were controlled by quorum sensing (16 activated, 14 repressed, >2-fold) when comparing the wild-type strain with the QS signal synthase and receptor mutants. Goal 3. We tested but did not identify a role for QS in B. japonicum in nodulation of soy bean plants. We also investigated whether the QS system identified in the B. japonicum type-strain (USDA110), bjaI-bjaR, was present in other Bradyrhizobium strains. In all of the B. japonicum strains (n=8) tested, we detected the production of the QS signal, isovaleryl-homoserine lactone. In addition, the genome sequences of other Bradyrhizobium strains are now available, and in most cases the QS genes bjaI-bjaR are present, suggesting that the QS system is important to the B. japonicum lifestyle. However we were unable to establish how it is important.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
Lindemann, A., G. Pessi, A. L. Schaefer, M. E. Mattmann, Q.H. Christensen, A. Kessler, H. Hennecke, H. E. Blackwell, E. P. Greenberg and C. S. Harwood. 2011. Quorum sensing in the soybean root-nodulating bacterium Bradyrhizobium japonicum: identification of isovaleryl-homoserine lactone, an unusual branched-chain signal. Proc. Natl. Acad. Sci. USA. 108:16750-16770.
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