Progress 07/15/07 to 12/31/11
Outputs
OUTPUTS: Presentations at meetings, workshops, seminars Teplitski, M. (Workshop) A quick guide to navigating your first job and your first real problems. Southern University and A&M College System. Nov 9, 2011. Baton Rouge, LA Teplitski, M. (Workshop) To compete or not to compete: can the Blue Ocean strategy be applied in an academic setting 97th Annual Meeting of Southeastern Branch of American Society for Microbiology, Gainesville, FL. October 20-22, 2011. Yagnik, K., M. Teplitski and M. Gao. Complementation study of expG gene in Sinorhizobium meliloti (Poster Presentation) 97th Annual Meeting of Southeastern Branch of American Society for Microbiology, Gainesville, FL. October 20-22, 2011. Teplitski, M. Cell-to-cell signaling and signal interference in host-associated bacterial communities. Biology Department, Florida State University, Tallahassee, October 13, 2011. Teplitski, M. Cell-to-cell signaling and signal interference in host-associated bacterial communities. UF Chemistry Department. September 9, 2011 Teplitski, M. (Seminar) Microbial Ecology, Ecosystem Health, Food Safety. Microbiology Graduate Student Symposium. Jacksonville, FL. August 21, 2010. Teplitski, M. (Presentation) The search for eukaryotic inhibitors of bacterial signaling: an update. Co-hosted by Smithsonian Marine Station and Harbor Branch Oceanographic Institute. Ft. Pierce, FL. July 17, 2008. Teplitski, M. (Oral Presentation) RIVET analysis of the in vivo regulation of symbiotically-relevant genes in Sinorhizobium meliloti. 20th North American Symbiotic Nitrogen Fixation Conference. July 10-14, 2007. Milwaukee, WI. Teplitski, M. (Lecture) Underground and underwater communications: who listens when microbes talk Florida Integrated Science Center USGS/USF. St. Petersburg, FL. August 15, 2007. Gao, M., Bauer, W.D., and Teplitski, M. Quorum sensing regulatory hierarchies in Sinorhizobium meliloti and their role in symbiosis. 20th North American Symbiotic Nitrogen Fixation Conference. July 10-14, 2007. Milwaukee, WI. Teplitski, M. (Invited Oral Presentation) Eukaryotic signal-mimics disrupt quorum sensing-dependent gene regulation in bacteria. Annual Meeting of American Society for Microbiology (Southeastern Branch). St. Pete Beach, October 27-29, 2005. PARTICIPANTS: Dr. Mengsheng Gao (Assistant In), Dr. Sathish Rajamani, Dr. Kanchan Bhan, Kruti Yagnik (undergraduate), Ali Alagely (undergraduate) TARGET AUDIENCES: Scientists, specifically those interested in host-bacterial interactions PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Accomplishments: 1. TnpR recombinase-based in vivo expression technology (RIVET) was adapted for S. meliloti. RIVET is at least as sensitive as GUS for documenting rhizobial gene regulation. In S. meliloti, RIVET has a unique application in that it allows to document gene regulation in un-differentiated rhizobia that live inside nodules. RIVET experiments demonstrated that the AHL synthase sinI was expressed in the rhizosphere and in the saprophytic rhizobia inside nodules (Teplitski and Gao, 2008). 2. Lumichrome, a derivative of the vitamin riboflavin, was shown to affect QS-dependent gene regulation in AHL reporters based on the LasR QS of P. aeruginosa. Studies with mutated reporters indicate that the effects of lumichrome on QS required a functional AHL receptor LasR (Rajamani et al., 2009). There appear to be multiple pathways leading to lumichrome synthesis in S. meliloti. In addition to the pathways that are homologous to those of E. coli, S. meliloti appears to have other cryptic pathways for lumichrome synthesis (Bauer and Yurgel, unpublished). 3. Even though the genome of S. meliloti contains multiple LuxR homologues, only two of them (ExpR and SinR) appear to function as AHL receptors. 4. The sole AHL synthase SinI produces multiple AHLs, all with long chain AHLs. There appears to be specificity in the ability of S. meliloti to detect these different AHLs and respond to them. Specifically, expression of sinI appears to be stimulated by all four major AHLs it produces, however the expression of wggR (expG) is only triggered by one of the AHLs (Gao et al, in preparation). 5. QS controls surface spreading in S. meliloti. This spreading requires production of copious amounts of EPS-2, but it is distinct from swarming, in which cells become hyperflagellated (Gao et al, in preparation). Many of the discoveries described above are generally in line with what is observed in other systems. We were initially puzzled by the very modest overlap between the results obtained with proteomic and microarray studies. Upon further investigation, we learned that these differences can be explained not by the differences in technical approaches, but by the role of a global post-transcriptional regulatory protein Hfq in controlling quorum sensing cascades. Unlike gamma-proteobacteria, which have at least two post-transcriptional regulatory systems (mediated by CsrA and Hfq), alpha-proteobacteria appear to rely exclusively on Hfq. We learned that Hfq-mediated post-transcriptional regulation strongly controls sinI under the laboratory conditions and in planta. Even though Hfq is known for its global regulatory role, the in-frame mutant was only modestly reduced in growth, was fully capable of utilizing most of the commercially available carbon sources, but was defective in its ability to assimilate branched amino acids. These results indicate that Hfq plays an important role in controlling bacterial signaling and assimilation of branched amino acids.
Publications
- Teplitski, M., Merighi, M., Gao, M., Robinson, J. 2011. Integration of cell-to-cell signals in soil bacterial communities. Soil Biology, vol. 23. Biocommunications in soil micro-organisms,pp.369-402. G.Witzany (ed). Springer-Verlaag.
- Teplitski, M., and Rajamani, S.R. 2011. Nutrient and signal exchange in the interactions between soil algae and bacteria. Soil Biology, vol. 23. Biocommunications in soil micro-organisms, pp.413-426. G.Witzany (ed). Springer-Verlaag.
- Alagely, A., Rajamani, S., Teplitski, M. 2011. Luminescent reporters and their applications for the characterization of signals and signal-mimics that alter LasR-mediated quorum sensing. Methods Mol Biol 692:113-1130
- Teplitski, M., Mathesius, U., Rumbaugh, K.P. 2010. Perception and degradation of N-acyl homoserine lactone quorum sensing signals by mammalian and plant cells. Chem Rev. 111:100-116
- Gao, M., Barnett, M., Long, S., Teplitski, M. 2010. Role of the Sinorhizobium meliloti global regulator Hfq in gene regulation and symbiosis. Mol Plant Microbe Interact 23:355-36
- Rajamani S., Bauer, W.D., Robinson, J.B., Farrow III, J.M., Pesci, E.C., Teplitski, M., Gao, M Sayre, R.T. and Phillips, D. A. 2008. The vitamin riboflavin and its derivative lumichrome activate the LasR bacterial quorum sensing receptor. Mol. Plant Microbe Interact. 21:1184-1192.
- Gao, M. and Teplitski, M. 2008. RIVET-a tool for in vivo analysis of symbiotically relevant gene expression in Sinorhizobium meliloti. Mol. Plant Microbe Interact. 21:162-170.
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Progress 07/15/09 to 07/14/10
Outputs
OUTPUTS: Guest lectures/seminars at other universities or departments: 1. "Plant-bacterial interactions: from rhizosphere biology to food safety". North Florida Research and Education Center, Quincy, FL. October 26, 2010. 2. "Microbial Ecology, Ecosystem Health, Food Safety". Microbiology Graduate Student Symposium. Jacksonville, FL. August 21, 2010. 3. "Are there roles for bacterial small regulatory RNAs in plant-bacterial interactions". University of Delaware & Delaware Biotechnology Institute, Newark, DE. May 6, 2009. PARTICIPANTS: 1. Dr. Mengsheng Gao (at UF). Dr. Gao started as a technician, and was promoted to a non-tenure track faculty. She developed her own CRIS project 2. Dr. Sathish Rajamani (at UC-Davis). 3. Ali Alagely (at UF). Mr. Alagely's presentation at the annual meeting (2009) of American Society for Microbiology (Florida Branch) was recognized as the Best Presentation by an Undergraduate. Ali developed and submitted an NSF Graduate Fellowship application TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Our studies of bacterial quorum sensing and the integration of QS into other global regulatory cascades led us a to a discovery that QS is subject to post-transcriptional regulation through the RNA-binding protein Hfq.
Impacts
We continued our investigation of the cell-to-cell signaling (Quorum Sensing) in the rhizosphere interactions of Sinorhizobium meliloti with its plant hosts. We have developed dual-labeled reporters to probe the dynamics of QS in the rhizosphere. Red-fluorescent protein reporters (RFP) were constructed in promoters of the AHL synthase gene sinI and a QS-controlled gene with a function in cell division ftsZ2. The reporters were moved into five gfp-labeled QS mutant backgrounds (incapable of perceiving or generating QS signals). Confocal analysis demonstrates that QS takes place at various stages of the symbiosis, consistent with our previous RIVET data. We have also developed sensitive luminescent reporters for characterizing agonists and antagonists of bacterial quorum sensing. These reporters are particularly useful for defining potential mode of action of the natural products with QS modulatory activities.
Publications
- (1) Teplitski, M., Merighi, M., Gao, M., Robinson, J. 2011. Integration of cell-to-cell signals in soil bacterial communities. Soil Biology, vol. 23. Biocommunications in soil microorganisms, pp.369-402. G.Witzany (ed). ISBN 978-3-642-14511-7. Springer-Verlaag. (2) Teplitski, M., and Rajamani, S.R. 2011. Nutrient and signal exchange in the interactions between soil algae and bacteria. Soil Biology, vol. 23. Biocommunications in soil microorganisms, pp.413-426. G.Witzany (ed). ISBN 978-3-642-14511-7. Springer-Verlaag. (3) Alagely, A., Rajamani, S., Teplitski, M. 2011. Luminescent reporters and their applications for the characterization of signals and signal-mimics that alter LasR-mediated quorum sensing. Methods Mol Biol 692:113-1130 (4) Teplitski, M., Mathesius, U., Rumbaugh, K.P. 2010. Perception and degradation of N-acyl homoserine lactone quorum sensing signals by mammalian and plant cells. Chem Rev. DOI: 10.1021/cr100045m (5) Gao, M., Barnett, M., Long, S., Teplitski, M. 2010. Role of the Sinorhizobium meliloti global regulator Hfq in gene regulation and symbiosis. Mol Plant Microbe Interact 23:355-365
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Progress 07/15/08 to 07/14/09
Outputs
OUTPUTS: 1) Presented 2 seminars: Teplitski, M. (Seminar) Novel layers of signaling complexity in legume-Rhizobium symbioses. Department of Agronomy, UF. Spring 2008. Teplitski, M. (Seminar) Bacterial quorum sensing and plant QS signal mimics in the Medicago-Sinorhizobium symbiosis. Department of Plant Pathology, UF. Spring 2008 2) Results of the research were presented at an international meeting: Gao, M., Teplitski. RIVET analysis symbiotic regulation of quorum sensing gene in Sinorhizobium meliloti. Abstracts of the 8th European Nitrogen fixation Conference, Gent, Belgium. August 30 -Sept 3, 2008. 3) RIVET (recombinase-based in vivo expression technology) has been adapted for Sinorhizobium (cloning vector pVMG-TnpR GenBank ID EU232661). A broad-host range promoter-recombinase probe plasmid pVMGCRT85 ("portable RIVET reporter" GenBank ID EU232662), suitable for other rhizobia was also constructed and released. PARTICIPANTS: Dr. Mengsheng Gao (at the University of Florida). Initially contributed to the project as a staff scientist, in 3/08 was promoted to a research faculty position at the University of Florida. Dr. Gao has adapted the RIVET system for Sinorhizobium, constructed and screened a library of promoter probe reporters, confirmed their regulation by RT-PCR. Dr. Gao's salary is paid, in part, by this grant. Elizabeth Creary (at the University of Florida). Carries out experiments on the role of in vivo QS signaling in controlling bacteroid-specific gene expression (Obj. 1A and 2C). Dr. Sathish Rajamani (at the University of California -Davis). Has defined the ability of the interkingdom vitamin signal lumichrome to interact with AHL receptor-based reporters. Potential genes involved in lumichrome production and secretion in S. meliloti are been screened for using transposon mutagenesis and genomic library screening. Efforts are underway to study their functions in a heterologous host. Dr. Rajamani constructed promoter-rfp reporter plasmids for the in planta investigation of the dynamics of QS regulation. Dr. Rajamani's salary was paid, in large part, by this grant. He is currently a post-doc at Darthmouth Zeeshan Qamar (at the University of California-Davis) is a Junior Specialist who contributes to the project by helping to identify a potential role for QS in controlling production of vitamin QS signal-mimics (riboflavin and lumichrome) in S. meliloti. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
RIVET, a tool for the investigation of gene regulation during different stages of symbiotic interactions has been adapted for the Sinorhizobium meliloti-Medicago model symbiosis (Gao and Teplitski, 2008. Mol Plant Microbe Interact 21(2):162-70). Vectors for making RIVET reporters are released. A library of promoter-gus constructs has been constructed and screened in vitro. Approximately a dozen promoters, previously not known to be controlled by QS, were identified. Their regulation by QS signals was confirmed by RT-PCR. Unexpectedly, a putative small regulatory RNA was identified as QS regulated, suggesting that there exist other, novel layers of QS signal integration. A manuscript detailing these observations is currently in review. Responses of these reporters to plant signals and to the inter-kingdom QS signal-mimic lumichrome are being investigated (Gao, Bauer, Teplitski). Over a dozen promoter-rfp reporter plasmids in the QS-responsive promoters have been constructed (Rajamani, Bauer, Robinson). Their regulation during different stages of the symbiosis is now being investigated (Robinson) The role of lumichrome, riboflavin in bacterial QS regulatory signal integration have been characterized (Rajamani et al., 2008. Mol Plant Microbe Interact 21(9): 1184-1192) Regulation of the AHL synthase, sinI, and four LuxR-type regulators was tested in vivo and in vitro. The sinI gene appears to be expressed weakly during early stages of rhizosphere colonization, but is then up-regulated during later stages of the rhizosphere colonization. The sinI gene is expressed strongly inside the nodules. The putative AHL receptors Smc00877 and Smc00878 are expressed strongly inside the nodule and on agar plates. LuxR-type homologs Smc04032 and Smc00658 appear to be differentially regulated on agar plates and inside the nodules. Regulation of all four LuxR-type regulators seems restricted to different nodule zones, suggesting their potential function in spatial or temporal regulation of nodule physiology (Gao, Teplitski, Bauer). The manuscript detailing these observations is currently in review.
Publications
- Rajamani S., Bauer W.D., Robinson J.B., Farrow J.M., Pesci E.C., Teplitski M., Sayre R.T., Phillips D.A. 2008. The vitamin riboflavin and its derivative lumichrome activate the LasR bacterial Quorum-Sensing receptor. Mol Plant Microbe Interact 21: 1184-1192.
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Progress 07/15/07 to 07/14/08
Outputs
OUTPUTS: 1. The results of this on-going project were presented at: a) Smithsonian Marine Station & Harbor Branch Oceanographic Institute. Title: "The search for eukaryotic inhibitors of bacterial signaling: an update". Invited seminar. July 17, 2008 Ft. Pierce, FL. b) UF Department of Agronomy. Title: "Novel layers of signaling complexity in legume-Rhizobium symbioses". 2008Gainesville, FL.. c) 20th North American Symbiotic Nitrogen Fixation Conference. Title: "RIVET analysis of the in vivo regulation of symbiotically-relevant genes in Sinorhizobium meliloti". July 10-14, 2007. Milwaukee, WI. d) Lake Alfred Citrus Research and Educational Center. Plant quorum sensing signal-mimics and their role in bacterial gene expression in vivo. April 28, 2007 Lake Alfred CREC, FL. 2. The characterization of the QS circuitry in S. meliloti and the discovery that QS may be controlling nodule senescence in legumes has led to a project "Exploring the role of QS in delayed nodule senescence in pasture legumes" that was submitted (as a pre-proposal) to the SARE program. Mr. B. Kurtz (Kurtz and Sons Dairy, Live Oak, FL) is a co-investigator on the SARE pre-proposal 3. A RIVET tool was adapted for S. meliloti, appropriate vectors were constructed and released. Gao, M. and Teplitski, M. 2008. Cloning vector pVMGCRT85, complete sequence. Accession EU232662. GenBank, NCBI-NIH. Gao, M., Oke, V. and Teplitski, M. 2008. Cloning vector pVMG-TnpR. Accession EU232661. GenBank, NCBI-NIH. PARTICIPANTS: Collaborators: Jayne Robinson (University of Dayton) W.Dietz Bauer (University of California-Davis) Partner: Nitya Jacob (Oxford College of Emory University) Investigators involved: Sathish Rajamani (post-doc), UC-Davis Mengsheng Gao (research faculty), University of Florida TARGET AUDIENCES: 1. Other academic scientists working in the area of symbiotic signaling, quorum sensing 2. Also a partnership with a local pasture/dairy operation was initiated in order to test variation in QS in native rhizobia in pasture legumes and identify a strain of rhizobia with delayed senescence. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In a screen for promoters controlled by quorum sensing (QS) in Sinorhizobium meliloti, a nitrogen-fixing endosymbiont of Medicago plants, genes involved in synthesis of symbiotically important exopolysaccharides (expG, expE, ndvA), DNA repair (uvrB, radA, xseA) and transport of metabolites (agpA, Smb20718, Smb21438) were identified. A putative transposase encoded by Sma2171 and a transcript found within the Sma1919 - Sma1921 intergenic region were also subject to QS regulation. Smc00877 and Smc00658, predicted LuxR homologues, contributed to the regulation of QS genes in Sinorhizobium, although it is not yet clear whether this regulation is direct. Production of N-acyl-homoserine lactone QS signals was reduced in the Smc00658 mutant, but not in the Smc00877 or Smc00878 mutants. Patterns of the in vitro and in planta expression of luxR homologues encoded by Smc00658, Smc00877, Smc00878 and Smc04032 suggest that they may be involved in gene regulation during different stages of the symbiosis, although signals perceived by these LuxR homologues are not yet known. Unlike typical LuxR AHL receptors, none of these LuxR homologues is located next to an AHL synthase. Based on their role in QS regulation and on the lack of association with an AHL synthase, these ORFs are designated as "orphan" LuxR homologues.
Publications
- Peer-reviewed publications: 1. Published Gao, M., Teplitski, M. 2008. RIVET - a tool for in vivo analysis of symbiotically relevant gene expression in Sinorhizobium meliloti. Mol Plant Microbe Interact: 21(2):162-70. 2. Accepted S. Rajamani, W. D. Bauer, J. B. Robinson, J. M. Farrow III, E. C. Pesci, M. Teplitski, M. Gao, R. T. Sayre, and D. A. Phillips. 2008. The vitamin riboflavin and its derivative lumichrome activate the LasR bacterial quorum sensing receptor. Molecular Plant Microbe Interact 3. Submitted: Gao, M., Teplitski, M. 2008. Contribution of "orphan" LuxR homologues to quorum sensing in Sinorhizobium meliloti. Submitted to J. Bacteriology
- Proceedings: Gao, M. and Teplitski, M. RIVET analysis of the in vivo regulation of symbiotically-relevant genes in Sinorhizobium meliloti. Abstracts of the 20th North American Symbiotic Nitrogen Fixation Conference. July 10-14, 2007. Milwaukee, WI. Gao, M., Bauer, W.D., and Teplitski, M. Quorum sensing regulatory hierarchies in Sinorhizobium meliloti and their role in symbiosis. Abstracts of the 20th North American Symbiotic Nitrogen Fixation Conference. July 10-14, 2007. Milwaukee, WI.
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