Objective 1. Identify genes in Sinorhizobium meliloti that are regulated by Quorum Sensing (QS) during symbiotic interactions with its plant host Medicago truncatula. Technically, this Objective will be accomplished in three steps: <BR> 1.1) identification of the in vivo QS regulon using RIVET screen <BR>1.2) expression analysis of the QS regulated genes during different stages of symbiosis (candidate genes will be selected based on RIVET screen results, and also on the results of our proteomic studies, and in vitro promoter-trap screening (Gao et al., unpublished)<BR> 1.3) interesting genes regulated by QS and/or AHL-mimics will be disrupted, and their symbiotic behavior will be tested. <BR><BR>Objective 2. Test the role of QS and host AHL mimics in the timing of symbiotic steps. Because we hypothesize that QS and plant AHL-mimics contribute to the precise timing of the symbiotic events, mutants which are turned ON or OFF at the wrong time or are insensitive to AHLs or plant signals may also prove informative in understanding the role of QS in the symbiosis. By studying the symbiotic behavior of these mutants, we may learn - for example - that QS is important to controlling proliferation of rhizobia inside the infection thread or nodules. QS may prove central to preventing rhizobia from rupturing host cells and becoming a pathogen. These hypotheses will be tested in three steps:<BR> 2.1) QS mutants with altered timing of their expression will be generated, and their behavior in planta will be studied <BR>2.2) the role of host AHL mimics and other host signals in manipulating bacterial QS genes will be assayed. Interesting novel host compounds will be purified. <BR>2.3) disruption of the host AHL-mimic synthesis will be attempted. <BR><BR>Objective 3. Identification of other bacterial regulatory pathways subject to manipulation by eukaryotic signals. Control over the interactions of bacteria with their eukaryotic hosts is not limited to QS. Recent high-throughput screens confirmed that other regulators (e.g. two component regulatory system GacS/GacA), as well as novel, uncharacterized genes are required for the interaction of bacteria with their hosts. Learning about the role of the gacS/gacA homologues in the pathogenesis may help to control many devastating and economically important diseases. For example, gacS, gacA homologs are found in Xylella spp and Xanthomonas spp, although their functions in disease are not yet known. Xylella and Xanthomonas pathovars cause citrus diseases (variegated chlorosis and citrus canker, respectively) with the potential to devastate Florida citrus culture.
NON-TECHNICAL SUMMARY: In natural and anthropogenic ecosystems, microbial communities play important roles in nutrient cycling, promoting plant growth, and in the removal/sequesteration of toxins and xenobiotics. Bacterial diseases of plants, animals and humans cause millions of dollars in damages. Based on the CDC estimates, over 4 billion cases of diarrhea caused by water- and food-borne bacteria claim lives of over 2 million people worldwide. In Florida, bacterial pathogens seriously threaten the quality of beaches and recreation areas, citrus and seafood industries. Tourism industry in FL is valued at $50 billion, citrus industry is at $8.5 bln, and the output of the seafood industry is $1.2 bln. Host-associated beneficial bacteria impact Florida economy as well. Beneficial bacteria promote healthy rhizosphere communities and fix atmospheric dinitrogen for both legumes and grasses. The ability to efficiently fix atmospheric nitrogen and supply it to the crops will reduce the need for N fertilizers. Based on the estimates of the Florida Department of Agriculture and Consumer Services, approximately 220,000 tons of Nitrogen-containing fertilizers are applied annually. Plant-associated nitrogen fixing bacteria can provide at least 40% of this nitrogen to their host plants at the "right" time in a "slow release" form. The goal of this study is to understand the role of bacterial cell-to-cell communication in the structuring of host-associated microbial communities. We aim to identify mechanisms that eukaryotes use to interfere with bacterial signaling.
APPROACH: Our primary approach will be to identify QS-regulated genes in planta using a modified RIVET screen. A positive selection RIVET based on the wild type resolvase cassette will be used. In this screen, the activation of a promoter-resolvase fusion will confer resistance to a selectable agent (fusaric acid) by resolving the genetic marker, res-tetRA-res at the res sites. A RIVET library of ~1-2 kB segments of S. meliloti genome will be cloned upstream of a promoterless resolvase gene. The library will be then transformed into the res-tetRA-res-marked S. meliloti wild type and into one of the already constructed mutants lacking a specific AHL receptor (expR, sinR) or AHL synthase (sinI). Pools of transformants will then be inoculated onto M. truncatula seedlings and then recovered from roots on a selective medium. Only the transformants in which the tetRA marker was resolved will grow on the selective medium, which contains zinc and fusaric acid. The selection will, therefore, tentatively identify those genes that were expressed in planta during the initial stages of symbiosis. These RIVET clones will then be recovered by single-step conjugative cloning. To identify the clones, they will be labeled and hybridized to the Sinorhizobium microarrays as described by Badinarayana et al. To identify bacteroid-specific QS regulated genes, we propose to take advantage of the already constructed and screened Okes library of ~ 100 bacteroid-specific promoter probes. We will screen this library for a subset that may be regulated by QS and host mimics. However, logically and mechanically Okes library and our proposed RIVET library are distinct. To further test the in vivo role of QS and plant compounds in controlling these genes, corresponding gfp fusions will be constructed. To characterize the role of host signals in the regulation of QS genes, we will extract compounds from freeze dried root exudates and roots of Medicago truncatula, fractionate them on HPLC and detect substances that activate or inhibit regulation of QS genes. Disrupting the ability to produce or secrete AHL-mimics is a logically necessary, though technically difficult experiment. Our earlier attempt to screen an M2 library of M. truncatula seedlings for mutants with altered AHL mimic production/secretion was not successful. As an alternative approach, we propose to use bacterial AiiA lactonase gene to construct functional mimic-deficient transgenics plants. The ability of plants to produce compounds that alter GacS-mediated signaling was first demonstrated by Koch et al., 2002, however the mechanism of this inhibition is now yet clear. We plan to screen HPLC fractions of plant exudates for the fractions which inhibit or activate expression of the csrB reporters. To identify genes which may contribute or disrupt GacS/GacA dependent regulation, we will screen metagenomic bacterial libraries for the ability of fosmid clones to affect the csrB reporter. These experiments will be technically similar to a screen of Williamson et al., 2005, who successfully identified metagenomic clones which alter QS-mediated gene expression
PROGRESS: 2006/10 TO 2007/09<BR>
OUTPUTS: Radio interview: Teplitski, M. Interview with Mid-Florida Public Radio on E. coli contamination of vegetable produce. Aired on September 18, 2006 Invited lectures/presentations: Survival of human pathogens in drinking water biofilms. Presented at the Annual Meeting of Florida Section of American Water Works Association. Orlando, Fl. November 27, 2006 Waterborne pathogens. Presented at Extension Symposium/Agent Training in Biosecurity and Food Safety. Gainesville, Fl. May 16, 2006 Plant quorum sensing signal-mimics and their role in bacterial gene expression in vivo. Pre- sented at the CREC "Plant Pathology and Friends" Seminar. April 28, 2006 Underground Communication: who listens when bacteria talk? Presented at Oxford College, Oxford, GA. April 2006 Bacterial quorum sensing in rhizosphere communication. Presented at Jacksonville State University, Jacksonville, AL. April 2006 Role of bacterial signaling in the structuring of host-associated microbial communities. Presented at Whitney Laboratory for Marine Bioscience, St. Augustine, Fl. March 10, 2006. <BR>PARTICIPANTS: Max Teplitski (PI) post-doctoral scientists: Mengsheng Gao and Jason Noel graduate students: Clayton Cox, Cory Krediet, Stephanie Halbig undergraduate students: Ali Al-Agely, Kiran Joglekar, Kush Bhorania middle/high school students: Emily Norman, Jennifer Kizza, Gaochuan Xie partners: Mote Marine Laboratory, University of California - Davis, Sidney Kimmel Cancer Center <BR>TARGET AUDIENCES: Individuals: post-doctoral scientists: Mengsheng Gao and Jason Noel -- each 10% graduate students: Clayton Cox, Cory Krediet, Stephanie Halbig -- each 5% undergraduate students: Ali Al-Agely, Kiran Joglekar, Kush Bhorania -- each 1% middle/high school students: Emily Norman, Jennifer Kizza, Gaochuan Xie -- each 1%
IMPACT: 2006/10 TO 2007/09<BR>
This research has led to several fundamental discoveries: 1) Experiments on the study of Salmonella in produce has led to several important discoveries. First, to test whether root colonization actually leads to spread of the pathogen into aboveground parts, a "printing technique" was employed for quick identification of bacteria inside plant organs. These results suggest that Salmonella 14028 moves into the aboveground parts of tomato, similar to observations in other plants. Although the mechanism of spread is not yet clear, individual Salmonella cells were present in apical meristems of tomatoes just three days after infection with as few as 100 cells per 1 ml of the hydroponic medium. Systemic infection of tomato stems with Salmonella has been shown to lead to fruit contamination under laboratory conditions (Guo et al., 2002). The ability of the strain 14028 to systemically colonize tomatoes is consistent with its competitiveness as an alfalfa endophyte. To begin learning about genes that are required for salmonella contamination of produce, we are screening a library of gfp promoter probe constructs. Further studies on this subject will lead to the improved food supplies. 2) Colonization of plants by bacteria requires a fine-tuned signal exchange between the two partners. To learn about the contribution of this multilevel QS interplay between plants and the associated bacteria, a promoter-probe library of S. meliloti is being constructed for recombinase-based in vivo expression (RIVET) assays. The characterization of bacterial genes regulated by QS during different stages of the symbiosis has already begun. Recombinase-based in vivo expression technology (RIVET) has been adapted for S. meliloti. Bicistronic promoterless tnpR-gus reporters were constructed to track expression of symbioti-cally-relevant S. meliloti genes during different stages of the interaction. Using RIVET, we have demonstrated that the regulation of cell division genes in un-differentiated sinorhizobia inside the nodules is distinct from what is known about cell division in bacteroids. Rhizosphere expression of the AHL synthase sinI::tnpR-gus reporter was modest in pre-quorate microcolonies, and then increased as cells within colonies reached quorum. AHL synthase and an AHL-regulated gene expG were activated inside the nodules. These preliminary results suggest that during plant colonization, host-bacterial signal exchange is fine-tuned, plant and bacterial signals play impor-tant roles at different stages of plant colonization by beneficial bacteria. Differences in symbi-otic regulation of bacterial cell division may suggest clues about evolution of the symbiosis.This is the first example of the global role of a pro-vitamin as a QS signal-mimic, and the first demonstration of a global role of a pro-vitamin in globally affecting bacterial QS gene expression.