Progress 02/01/10 to 01/31/14
Outputs
Target Audience: Target audiences of agricultural scientists were reached by publication (see Publications) and research presentations (see Activities) Changes/Problems: 2 no-cost extensions were granted. Project goals were achieved with the extended time. What opportunities for training and professional development has the project provided? 1 postdoctoral scholar, 1 graduate student, one early-career technician and 2 undergraduate students were trained. How have the results been disseminated to communities of interest? Presentations: Jones, K. M. (2013, April). The role of bacterial exopolysaccharides in the invasion of legume plant hosts by rhizobial symbionts. Delivered at University of North Texas, Department of Biology. (National) Jones, K. M. (2012, August). The role of exopolysaccharides in the invasion of legume plant hosts by bacterial symbionts. Delivered at University of Florida, Department of Plant Pathology Seminar. (National) Jones, K. M. (2011, September). The role of rhizobial acidic exopolysaccharides in symbiotic colonization of plant hosts. Delivered at FSU/FAMU Department of Chemical and Biomedical Engineering. (National) Jones, K. M. (presented 2013, December). The role of Sinorhizbium meliloti succinoglycan in Medicago host plant invasion. Presentation at Medicago truncatula Resources Workshop, The Samuel Roberts Noble Foundation, The Samuel Roberts Noble Foundation, Ardmore, OK. (International) Jones, K. M. (presented 2013, November). The role of the bacterial polysaccharide succinoglycan in Medicago host plant invasion by Sinorhizobium meliloti. Presentation at The 99th Annual Meeting, Southeastern Branch of the American Society for Microbiology, Auburn University, Auburn AL. (Regional) Jones, K. M. (presented 2013, July). The role of the bacterial polysaccharide succinoglycan in Medicago host plant invasion by Sinorhizobium meliloti. Presentation at 22nd North American Symbiotic Nitrogen Fixation Conference, Organizing committee, University of Minnesota, Minneapolis, MN. (International) Jones, K. M. (presented 2012, July). The role of exopolysaccharides in the invasion of legume plant hosts by bacterial symbionts. Presentation at NRI/AFRI Microbial Biology and Microbial Functional Genomics Awardee Meeting, NRI/AFRI US Department of Agriculture, Washington, D. C. (National) Jones, K. M. (presented 2011, December). Symbiosis and senescence of nitrogen-fixing rhizobial bacteria within host plant cells. Presentation at FRONTIERS IN MACROMOLECULAR IMAGING, Florida State University, Tallahassee, FL. (International) Jones, K. M., Davis, O. M., Mendis, H. C., & Washburn, B. K. (presented 2011, July). The role of exopolysaccharides in the invasion of legume plant hosts by bacterial symbionts. Poster presentation at NRI/AFRI Microbial Biology and Microbial Functional Genomics Awardee Meeting, US Department of Agriculture, Washington, D. C. (National) Queiroux, C., Washburn, B. K., Davis, O. M., Brewer, T. E., Stewart, J., Lyons, M., & Jones, K. M. (presented 2011, May). Rhizobial factors that promote symbiotic nitrogen fixation, identified by a comparative genomic approach. Poster presentation at American Society for Microbiology, General Meeting, American Society for Microbiology, New Orleans, LA. (National) What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
S. meliloti exoK deletion mutants lacking only the succinoglycan endoglycanase encoded by exoK have only a minor defect in symbiosis We have shown that previously-isolated exoK::Tn5 mutants deficient in the exoK glycanase have a symbiotic defect on both alfalfa and M. truncatula, but that the defect is much more severe on M. truncatula than on alfalfa. This mutant is impaired in its ability to convert HMW succinoglycan to LMW. Our further analysis has shown that constitutive expression of the exoK glycanase alone from the pExoK::RF771 plasmid does not complement the symbiotic defect of an exoK::Tn5 mutant, but the pEX154 cosmid that also contains the downstream exoLAMON operon does complement the defect. Expression of exoK alone on the pExoK::RF771 plasmid is sufficient to complement production of LMW succinoglycan in the exoK::Tn5 mutant as demonstrated in a “Calcofluor-halo assay”. (Detection of succinoglycan by staining with the fluorescent brightener Calcofluor White M2R.) This assay shows that Calcofluor-fluorescing succinoglycan is being made in a LMW form that can diffuse away from S. meliloti cells growing on agar medium and produce a fluorescent halo. These results suggested that the symbiotic phenotype of the exoK::Tn5 mutant was not due to the loss of exoK alone. We suspected that this might be due to a negative polar effect on expression of the downstream exoLAMON genes in the exoK::Tn5 mutant. qPCR tests confirmed that, as we suspected, the exoK::Tn5 mutant had reduced expression of the downstream gene exoL. Thus the existing exoK::Tn5 mutant was not adequate for determining the role of LMW succinoglycan in infection. To determine the phenotype of a true loss-of-function exoK mutant, we constructed new mutants in which the loss of exoK function could be separated from negative polar effects on the exoLAMON operon. This series of strains was used to confirm that the polar effect on the exoLAMON genes accounted for the lack of complementation of exoK::Tn5 by expression of exoK alone and to provide mutants that are deficient solely in exoK expression. First, we constructed strains in which the exoHK genes are separated from the exoLAMON genes by a neomycin-resistance cassette and in which expression of the downstream exoLAMON genes is driven by the trp promoter (named “trpexoL” strains). These strains have no symbiotic defect. We also constructed strains isogenic with the trpexoL strains, except that exoK has been deleted, called Kdel strains. These strains are defective for symbiosis on M. truncatula and on alfalfa, but the defect is not as severe as that of the exoK::Tn5 mutant. Unlike the exoK::Tn5 mutant, the symbiotic defect of the Kdel mutants can be complemented by expression of exoK alone, indicating that the Kdel deletion does not have a detrimental polar effect on downstream genes. The results of the Kdel mutant tests indicate that reduction in LMW succinoglycan levels due to loss of the exoK glycanase has only a moderate effect on symbiotic proficiency. Mutants lacking both the exoH-encoded succinyltransferase and the exoK-encoded endoglycanase completely lack symbiotic proficiency, in contrast to those lacking only the exoK-encoded glycanase As described above, exoK and exoH lie just upstream of the exoLAMON genes, and the previously isolated exoK::Tn5 mutant was shown to have polar effects on exoLAMON expression. Similarly, the exoH mutants analyzed in previous studies carry an internal transposon insertion that is expected to have negative polar effects on the downstream exoLAMON genes. In order to eliminate these polar effects and determine the phenotype of a true deletion of the exoH succinyltransferase, an approach similar to the construction of the exoK deletion in the Kdel strain (see above) was used. We have now constructed strains that are isogenic with the Kdel strains, except that the exoH gene is also deleted. There is a profound difference in the invasion deficiency of the exoK exoH double deletion mutants (called HKdel) compared to the exoK deletion strains (Kdel). As described above, the HKdel mutants have the exoLAMON genes under trp promoter control, eliminating the negative polar effect on their expression. Thus, the HKdel strains differ from the Kdel strains only in the lack of the exoH succinyltransferase. After 9 weeks the HKdel mutant inoculated plants were still no more successful than uninoculated plants. Thus succinylation by exoH appears to be much more important for successful symbiosis than cleavage to the LMW form by exoK. It should be noted that since exoH lies immediately upstream of exoK, the exoH::Tn5 mutant is likely to also be defective for exoK functions. However, in vitro tests of ExoK enzyme function in a previous study indicate that the unsuccinylated succinoglycan produced by the exoH::Tn5 mutant is uncleavable by ExoK, so the phenotype of the HKdel mutant is expected to be unaltered by the presence of an exoK-complementing plasmid. This assumption is being verified. Production of LMW succinoglycan by the exsH-encoded endoglycanase makes no contribution to S. meliloti symbiotic proficiency It has previously been demonstrated that the unsuccinylated succinoglycan produced by an exoH::Tn5 mutant is resistant to glycanase cleavage, and produces only 5% of its succinoglycan in the LMW form, compared with ~50% in the WT. It has been speculated that strains lacking the exoH succinyltransferase are therefore deficient for host invasion because they lack the LMW fraction of succinoglycan. However, as described above, mutants lacking the exoK glycanase do not have as severe a symbiotic defect as mutants lacking the exoH succinyltransferase. One possible explanation for the observation is that other glycanases may be able to process wild type succinoglycan, but not succinyl-deficient succinoglycan, to the LMW form. In fact, another glycanase called ExsH (a glycosyl hydrolase family 16 laminarinase) has also been shown to cleave succinoglycan in vitro and in vivo. An exoK::Tn5-233/exsH::Tn5 double mutant was constructed in this earlier study, however, its symbiotic phenotype was not quantified in alfalfa, or tested in M. truncatula. We have explored the role of exsH as described below. We have constructed an exsH null mutant that carries an internal insertion of the transposon Tn5-233. Polar effects of this transposon insertion are not a concern for this mutant because the genes downstream of exsH are encoded on the opposite strand. We have now made the exsH double mutant series with trpexoL, Kdel and HKdel. We have found that the phenotype of the Kdel/exsH::Tn5 double mutant, deficient for both known succinoglycan glycanases, is very similar to the intermediate phenotype of the Kdel single mutant. (The control strain trpexo/exsH has no symbiotic defect.) Since knockout of exsH has little if any effect on symbiosis, this suggests that the ExsH glycanase cannot fulfill functions of the ExoK glycanase during infection thread formation. The HKdel/exsH::Tn5 we constructed will also be tested, and is expected to have a severe symbiotic defect similar to the HKdel mutant. Consistent with the result that knockout of exsH results in no symbiotic defect, we have preliminary evidence showing that an exsH::b-glucuronidase (GUS) reporter fusion is not expressed at all by S. meliloti on the plant surface, during invasion, or in nodules, although it is expressed by S. meliloti growing on agar plates. Despite the fact that exsH encodes a glycanase that can cleave succinoglycan in vitro and in free-living cells, this glycanase does not appear to be expressed during host invasion, nor does its absence appear to have any effect on host invasion.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Jones, K. M. (2012). Increased production of the exopolysaccharide succinoglycan enhances Sinorhizobium meliloti 1021 symbiosis with the host plant Medicago truncatula. Journal of Bacteriology, 194, 4322-4331.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Mendis, H. C., Queiroux, C., Brewer, T. E., Davis, O. M., Washburn, B. K., & Jones, K. M. (2013). The succinoglycan endoglycanase encoded by exoK is required for efficient symbiosis of Sinorhizobium meliloti 1021 with the host plants Medicago truncatula and Medicago sativa (Alfalfa). Mol Plant Microbe Interact, 26(9), 1089-1105.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Jones, K. M., Mendis, H. C., & Queiroux, C. (2013). Single-plant, sterile microcosms for nodulation and growth of the legume plant Medicago truncatula with the rhizobial symbiont Sinorhizobium meliloti. J Vis Exp, 80, 1-13.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Brewer, T. E., Stroupe, M.E., & Jones, K. M. (2014). The genome, proteome and phylogenetic analysis of Sinorhizobium meliloti phage ?M12, the founder of a new group of T4-superfamily phages. Virology, 450451(0), 84-97.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Stroupe, M. E., Brewer, T. E., Sousa, D. R., & Jones, K. M. (2014). The structure of Sinorhizobium meliloti phage ?M12, which has a novel T=19l triangulation number and is the founder of a new group of T4-superfamily phages. Virology, 450451(0), 205-212.
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