The shikimate pathway produces the precursors for a variety of essential effector compounds secreted by bacteria. One type of such compound, known as siderophores are essential for the acquisition of iron in environments where it’s limited. Thus the ability to secrete siderophores confers a competitive environment to bacteria that can secrete them and allows them to influence the makeup of their microbial communities. These are the major focus of this project, which examines how shikimate pathways derived secondary metabolite levels in rhizospheric soil affect the levels of siderophore secreting bacteria present, and how that consequently impacts the rhizosphere microbiome.

Figure 1: Electrophoretic mobility shift assay (EMSA) showing complexation of RifR with putative promoter on an increasing concentration gradient of A. thaliana root exudate. RifR protein concentration was held constant at 120nM (shown as present or absent by checkmarks and crosses respectively), as was the promoter probe concentration (at 12ng/μl).  Root exudate concentration is expressed in relative factors of the pooled collection prior to concentration. Assay was conducted usingbiotinilated PCR product of putative promoter and detected using streptavidin bound horse radish peroxidase (HRP) givenluminol as substrate.

For this project, Jilei invests her efforts to learn more about the involvement of the shikimate pathway and siderophores in the rhizosphere. Pseudomonas putida is an important bacteria known to colonize the roots of many commercial crops allowing for beneficial exchange of synthesized compounds1,2,3. Known for its metabolic versatility, this species has often been manipulated as part of efforts in bioremediation in addition to developments in biotechnology4,5. Our team has identified a novel IclR type regulator (pp_2609) in P. putida directly upstream of a functionally uncharacterized operon that we suspect to be involved rhizospheric interactions. This operon includes homologs of a RifI sub-class of shikimate dehydrogenases, an major facilitator superfamily (MFS) transporter, and an oxosteroid dehydrogenase among other dehydrogenases, which could together be involved in a new metabolic pathway in P. putida. The pp_2609 protein has been demonstrated to specifically bind to the target promoter of this operon and stabilize its binding in the presence of Arabadopsis thaliana root exudates (Figure 1) via electrophoretic mobility shift assays (EMSAs). While these findings are being confirmed using LacZ-promoter fusion experiments, the pp_2609 protein was also crystallized to determine its structure (Figure 2).  The 2Å resolution three dimensional structure of pp_2609 has been found to be structurally homologous to that of other IclR regulators and have helped identify the regulator’s putative metabolite binding pockets. Further studies in effector binding to this protein will also be conducted to gain insight into the metabolic role of its target operon, but we are reasonably confident that we have identified a new rhizospherically important transcriptional regulator in P. putida.

Figure 2: 3D model of RifR structure solved from X-ray crystallography (Space group: C121,Resolution: 40-1.95Å, Rfree 26.2, Rwork: 22.4). X-ray diffraction data was collected at the Advanced Proton Source (Argon, IL), Beamline 17-ID.  Protein diffracted as a tetramer fromits asymmetric unit. Tetramer is composed of two asymmetrical dimers, made up of twoidentical monomers that combine to form the DNA binding domains.

References:

1) Ongena M., Jourdan E., Adam A., Schäfer M., Budzikiewicz H., and Thonart P. 2008. Amino acids, iron, and growth rate as key factors influencing production of the Pseudomonas putida BTP1 benzylamine derivative involved in systemic resistance induction in different plants. Microb Ecol. 55(2):280-292. doi:10.1007/s00248-007-9275-5
2) Neal A.L., Ahmad S., Gordon-Weeks R., and Ton J. 2012. Benzoxazinoids in root exudates of maize attract pseudomonas putida to the rhizosphere. PLoS One. 7(4):e35498. doi:10.1371/journal.pone.0035498
3) Srivastava S., Chaudhry V., Mishra A., et al. 2012. Gene expression profiling through microarray analysis in Arabidopsis thaliana colonized by Pseudomonas putida MTCC5279, a plant growth promoting rhizobacterium. Plant Signal Behav. 2012;7(2):235-245.
4) Nagarajan K. and Loh K.C. 2014. Formulation of microbial cocktails for BTEX biodegradation. Biodegradation. 26(1):51-63. doi:10.1007/s10532-014-9715-0
5) Tropel D. and Meer J.R. Van Der. 2004. Bacterial Transcriptional Regulators for Degradation Pathways of Aromatic Compounds. Microbiol Mol Biol Rev. 68(3):474-500. doi:10.1128/MMBR.68.3.474