Progress 10/01/01 to 09/30/05
Outputs
Despite extensive study, little is known about what holds the organisms of the soil and rhizosphere together as a community. Better understanding of the function of community in this context may suggest strategies to improve the success of inoculants for crop production, because survival of the introduced bacteria in the rhizosphere community that must ultimately be managed successfully. We are studying regulation of genes in Bacillus cereus whose expression is affected by other members of the rhizosphere community. B. cereus provides an excellent model system for a study of gene regulation because there is a substantial body of knowledge and techniques for its study, but little is known about its gene regulation or communication with other organisms. We developed a promoter trap vector and fluorescent cell sorting method to isolate promoters responsive to environmental signals. With these powerful tools, we have identified a number of B. cereus genes that are
regulated by other rhizosphere inhabitants, including P. aureofaciens and Flavobacterium johnsoniae (Fj). We found that B. cereus stimulates the growth of Fj approximately 1000-fold in root exudate medium. This year we showed that the mechanism of growth promotion is that B. cereus provides Fj with peptidoglycan fragments that stimulate growth. We are also exploring the production of bacterial signals by both rhizosphere and soil communities. To access both readily cultured and unculturable bacteria, we are using metagenomics, or the analysis of the genomes of a group of organisms. We have constructed libraries containing more than 9 Gb of DNA from Alaskan soil and screened these libraries for production of small molecules that induce quorum sensing. The quorum sensing promoter is highly sensitive to small molecules and therefore acts as a broad sensor for biologically active compounds. We developed an intracellular assay, designated METREX, in which the same cell carries the
metagenomic clone and the biosensor that indicates whether the clone is directing the synthesis of a biologically active molecule. With this screen, we identified new compounds that induce quorum sensing as well as those that have antibiotic activity.
Impacts
The expected impact is broad. Bacillus cereus is a proven biocontrol agent for plant disease and therefore understanding its interactions in the rhizosphere will contribute to its wise deployment. The small molecules identified in the METREX screen have the potential to contribute to development of antibiotics, insecticides and other products that may have direct application in agriculture, both in crop health and animal health. Fundamental ecological understanding that may emerge from soil metagenomics will provide the basis for better management of soil nutrition and plant health.
Publications
- Peterson, S.B., Dunn, A.K., Klimowicz, A.K., and Handelsman, J. 2006. Peptidoglycan from Bacillus cereus mediates commensalism with rhizosphere bacteria from the Cytophaga-Flavobacterium group. Appl. Environ. Microbiol. (submitted).
- Williamson, L.L., Borlee, B.R., Schloss, P.D., Guan, C., Allen, H.K., and Handelsman, J. 2005. Intracellular screen to identify metagenomic clones that induce or inhibit a quorum-sensing biosensor. Appl. Environ. Microbiol. 71:6335-6344.
- Handelsman, J. 2005. How to find new antibiotics. The Scientist. 19:20. Schloss, P. D., and J. Handelsman. 2005. Introducing DOTUR, a computer program for defining operational taxonomic units and species richness. Appl. Environ. Microbiol. 71(3):1501-1506.
- Handelsman, J. 2005. Sorting out metagenomes. Nature Biotechnology. 23(1):38-39.
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Progress 01/01/04 to 12/31/04
Outputs
The health of plants derives from their relationships with the teeming community of microorganisms in the surrounding rhizosphere. These microorganisms communicate, cooperate and combat one another in a dynamic symbiosis with the host plant. Despite extensive study, little is known about what holds these dramatically interactive microorganisms together as a community. Better understanding of the function of community in this context may suggest strategies to improve the success of inoculants for crop production, because survival of the introduced bacteria in the rhizosphere community that must ultimately be managed successfully. We are studying regulation of genes in Bacillus cereus whose expression is affected by other members of the rhizosphere community. B. cereus provides an excellent model system for a study of gene regulation because there is a substantial body of knowledge and techniques for its study, but little is known about its gene regulation or
communication with other organisms. We developed a promoter trap vector and fluorescent cell sorting method to isolate promoters responsive to environmental signals. With these powerful tools, we have identified a number of B. cereus genes that are regulated by other rhizosphere inhabitants, including P. aureofaciens and Flavobacterium johnsoniae (Fj). One gene from Fj, which codes for a putative negative regulator, is being studied by mutant analysis. Moreover, we found that B. cereus stimulates the growth of Fj approximately 1000-fold in root exudate medium, and the mechanism of growth promotion is under investigation by genetic analysis. Preliminary evidence suggests that peptidoglycan fragments released by B. cereus stimulate Fj growth. Biochemical and genetic experiments are in progress to test this model. We also found that genes in B. cereus are regulated by homoserine lactones, the signaling molecules used by gram-negative bacteria to control population-dependent gene
expression. This is, to our knowledge, the first identification of homoserine lactone-regulated genes in a gram-positive organism. Finally, we are studying the role of a homoserine lactonase produced by B. cereus. We have constructed two mutants that carry insertions in aiiA, the gene encoding the lactonase, and we are examining the interaction of these mutants with members of the rhizosphere community that are known to use homoserine lactones for signaling. We have completed sequencing the B. cereus strain UW85 genome and found redundancy in signaling functions. We found two other candidate lactonases and are currently constructing knock-out mutants in each to determine their role in rhizosphere communication.
Impacts
Bacillus cereus is a proven biocontrol agent for root diseases of soybeans and alfalfa. Understanding the relationship between B. cereus and the microbial community in which it must function may lead to improved strategies for biocontrol of plant disease.
Publications
- Schloss, P. D., B. R. Larget, and J. Handelsman. 2004. Integration of microbial ecology and statistics: a test to compare gene libraries. Appl. Environ Microbiol. 70(9):5485-92.
- Emmert, E.A.B., Klimowicz, A.K., Thomas, M.G., and Handelsman, J. 2004. Genetics of zwittermicin A production in Bacillus cereus. Appl. Environ. Microbiol. 70: 104-113.
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Progress 01/01/03 to 12/31/03
Outputs
The health of plants derives from their relationships with the teeming community of microorganisms in the surrounding rhizosphere. These microorganisms communicate, cooperate and combat one another in a dynamic symbiosis with the host plant. Despite extensive study, little is known about what holds these dramatically interactive microorganisms together as a community. Better understanding of the function of community in this context may suggest strategies to improve the success of inoculants for crop production, because survival of the introduced bacteria in the rhizosphere community must ultimately be managed successfully. We are studying regulation of genes in Bacillus cereus whose expression is affected by other members of the rhizosphere community. B. cereus provides an excellent model system for a study of gene regulation because there is a substantial body of knowledge and techniques for its study, but little is known about its gene regulation or communication
with other organisms. We developed a promoter trap vector and fluorescent cell sorting method to isolate promoters responsive to environmental signals. With these powerful tools, we have identified a number of B. cereus genes that are regulated by other rhizosphere inhabitants, including P. aureofaciens and Flavobacterium johnsoniae (Fj). One gene from Fj, which codes for a putative negative regulator, is being studied by mutant analysis. Moreover, we found that B. cereus stimulates the growth of Fj approximately 1000-fold in root exudate medium, and the mechanism of growth promotion is under investigation by genetic analysis. We also found that genes in B. cereus are regulated by homoserine lactones, the signalling molecules used by gram-negative bacteria to control population-dependent gene expression. This is, to our knowledge, the first identification of homoserine lactone-regulated genes in a gram-positive organism. Finally, we are studying the role of a homoserine lactonase
produced by B. cereus. We have constructed two mutants that carry insertions in aiiA, the gene encoding the lactonase, and we are examining the interaction of these mutants with members of the rhizosphere community that are known to use homoserine lactones for signaling. We have completed sequencing the B. cereus strain UW85 genome and found redundancy in signaling functions. We found two other candidate lactonases and are currently constructing knock-out mutants in each to determine their role in rhizosphere communication. We are investigating the possibility that rhizosphere communication is mediated by zwittermicin A, the novel antibiotic we discovered in B. cereus. This year, we completed characterizing mutants in zwittermicin A biosynthesis and identified 10 genes involved. In the genome sequencing project, we recently found what appears to be the complete biosynthetic cluster, spanning 65 kb, on a single BAC clone. Expression of this cluster in E. coli and B. subtilis is
underway.
Impacts
Bacillus cereus is a proven biocontrol agent for root diseases of soybeans and alfalfa. Understanding the relationship between B. cereus and the microbial community in which it must function may lead to improved strategies for biocontrol of plant disease.
Publications
- Dunn, A.K., Klimowicz, A.K., and Handelsman, J. 2003. Use of a promoter trap to identify Bacillus cereus genes regulated by tomato seed exudate and a rhizosphere resident, Pseudomonas aureofaciens. Appl. Environ. Microbiol. 69: 1197-205.
- Emmert, E.A.B., Klimowicz, A.K., Thomas, M.G., and Handelsman, J. 2004. Genetics of zwittermicin A production in Bacillus cereus. Appl. Environ. Microbiol. 70: 104-113.
- Handelsman, J. 2003. Soils - the metagenomics approach. In: Microbial Diversity Bioprospecting. Alan T. Bull, ed. American Society for Microbiology Press. Broderick, N.A., Goodman, R.M., Handelsman, J., and Raffa, K.F. 2003. Effect of host diet and insect source on synergy of gypsy moth (Lepidoptera:Lymantriidae) mortality to Bacillus thuringiensis subsp. kurstaki by zwittermicin A. Environ. Entomol. 32: 387-391.
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Progress 01/01/02 to 12/31/02
Outputs
The health of plants derives from their relationships with the teeming community of microorganisms in the surrounding rhizosphere. These microorganisms communicate, cooperate and combat one another in a dynamic symbiosis with the host plant. Despite extensive study, little is known about what holds these dramatically interactive microorganisms together as a community. Better understanding of the function of community in this context may suggest strategies to improve the success of inoculants for crop production, because it is survival of the introduced bacteria in the rhizosphere community that must ultimately be managed successfully. Central to all organismal communication are chemical signals that influence gene expression. Despite extensive work exploring the nature and role of chemical signaling within populations of single species of microorganisms and between plants and microorganisms, such as Rhizobium and Agrobacterium, chemical signaling between species in
microbial communities has been largely ignored. Bacteria are expert at sensing their environments, and other bacteria represent a powerful aspect of the microbial environment; thus, it seems likely that they have systems for sensing and responding to each other's chemical signals. Elucidation of the mechanisms of chemical signaling is key to understanding the function of the rhizosphere community. We will study regulation of genes in Bacillus cereus whose expression is affected by other members of the rhizosphere community. B. cereus provides an excellent model system for a study of gene regulation because there is a substantial body of knowledge and techniques for its study, but little is known about its gene regulation or communication with other organisms. We developed a promoter trap vector and fluorescent cell sorting method to isolate promoters responsive to environmental signals. With these powerful tools, we have identified a number of B. cereus genes that are regulated by
other rhizosphere inhabitants, including P. aureofaciens. We also found that genes in B. cereus are regulated by homoserine lactones, the signalling molecules used by gram-negative bacteria to control population-dependent gene expression. This is, to our knowledge, the first identification of homoserine lactone-regulated genes in a gram-positive organism. Finally, we are studying the role of a homoserine lactonase produced by B. cereus. We have constructed two mutants that carry insertions in aiiA, the gene encoding the lactonase, and we are examining the interaction of these mutants with members of the rhizosphere community that are known to use homoserine lactones for signaling.
Impacts
Bacillus cereus is a proven biocontrol agent for root diseases of soybeans and alfalfa. Understanding the relationship between B. cereus and the microbial community in which it must function may lead to improved strategies for biocontrol of plant disease.
Publications
- Dunn, A.K., and Handelsman, J. 2002. Toward an understanding of microbial communities through analysis of communication networks. ISME 9 Antonie von Leeuwenhoek 81: 565-574.
- Handelsman, J., and Wackett, L. 2002. Ecology and industrial microbiology: Microbial diversity - sustaining the Earth and industry. Current Op. Microbiol. 5:237-239.
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Progress 01/01/01 to 12/31/01
Outputs
The health of plants derives from their relationships with the teeming community of microorganisms in the surrounding rhizosphere. These microorganisms communicate, cooperate and combat one another in a dynamic symbiosis with the host plant. Despite extensive study, little is known about what holds these dramatically interactive microorganisms together as a community. Better understanding of the function of community in this context may suggest strategies to improve the success of inoculants for crop production, because survival of the introduced bacteria in the rhizosphere community that must ultimately be managed successfully. Central to all organismal communication are chemical signals that influence gene expression. Despite extensive work exploring the nature and role of chemical signaling within populations of single species of microorganisms and between plants and microorganisms, such as Rhizobium and Agrobacterium, chemical signaling between species in
microbial communities has been largely ignored. Bacteria are expert at sensing their environments, and other bacteria represent a powerful aspect of the microbial environment; thus, it seems likely that they have systems for sensing and responding to each other's chemical signals. Elucidation of the mechanisms of chemical signaling is key to understanding the function of the rhizosphere community. We will study regulation of genes in Bacillus cereus whose expression is affected by other members of the rhizosphere community. B. cereus provides an excellent model system for a study of gene regulation because there is a substantial body of knowledge and techniques for its study, but little is known about its gene regulation or communication with other organisms. We developed a promoter trap vector and fluorescent cell sorting method to isolate promoters responsive to environmental signals. With this system, we have identified two genes from B. cereus that are repressed by cultures of
another rhizosphere bacterium, Pseudomonas aureofaciens. In the proposed project, we will identify other B. cereus sequences regulated by P. aureofaciens, Cytophaga johnsonae, Bradyrhizobium japonicum, Pythium torulosum, and a collection of rhizosphere bacteria. We will then use a mutational analysis to determine the role of the genes that are contiguous with these sequences in B. cereus and identify the factors in the bacterial cultures that influence the expression of the B. cereus genes. The initial stages of the work have been devoted to characterizing phylogenetic identification of bacteria that co-isolate with B. cereus from field-grown plants, which will be candidates for screening for induction of B. cereus genes. We found that 3 to 5% of the B. cereus isolates carry cryptic contaminants that survive multiple colony purifications and only become visible to the eye after the B. cereus cultures have been incubated at 4 C for 14 days. The organisms identified thus far contain 16S
rRNA sequences that align with members of the Pseudomonas, Cytophaga, and Variovorax genera.
Impacts
Bacillus cereus is a proven biocontrol agent for root diseases of soybeans and alfalfa. Understanding the relationship between B. cereus and the microbial community in which it must function may lead to improved strategies for biocontrol of plant disease.
Publications
- No publications reported this period
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