The plant pathogenic bacterium Pantoea stewartii subsp. stewartii (P. stewartii) produces an exopolysaccharide (EPS) virulence factor that plays a major role in the cause of Stewart's wilt disease in maize. The EPS biosynthetic functions are encoded by the cps gene system, which is expressed in a cell density-dependent manner governed by quorum sensing regulation. Embedded within this biosynthetic locus are two genes, wceF and wceJ, whose enzyme products have a role in the measured degradation of the secreted glycopolymer. The preliminary data for this project support the hypothesis that WceF is an EPS hydrolase, and WceJ an EPS modifying enzyme, which transforms the EPS polymer into a substrate suitable for WceF-specific EPS depolymerization. Thus, the specific objectives for this project are to experimentally define and verify the enzymatic and apparent cooperative roles of WceF and WceJ as EPS degradative functions and to chemically characterize the EPS degradative products. Additionally and importantly, this project seeks to determine the biological significance of coordinate stewartan EPS synthesis and depolymerization. The preliminary data indicate that these functions play a major role in the Stewart's wilt infection process. The project focuses on a newly recognized bacterial EPS metabolic dynamic that plays a major role in pathogenesis. This novel understanding may reveal new opportunities for disease control. In addition, this project provides excellent training opportunities postdoctoral fellows and students at all levels.
Federal Award ID: Award 1053869 in the amount of $72,986 was a supplement to Award 0619194 Report Submission Period: 07/01/2011 to 09/30/2011 Intellectual Merit: Supplement 1053869 was to complete critical experiments aimed at resolving the complex stewartan and amylovora exopolysaccharide (EPS) biosynthetic pathways in two related plant pathogenic bacteria. Pantoea stewartii subsp. stewartii is the causal agent of corn Stewarts vascular wilt, and E. amylovora is the cause of fire blight disease in apple and pear. The objective was to resolve inconsistencies in the functional assignments of the genes encoded by the two related biosynthetic pathways, and define, in particular, two genes that appeared to be non-essential for the production of stewartan EPS. The comparative genetic and EPS structural analyses were a necessary expansion of the original project focus because the results of cross-functionality experiments were needed for the unequivocal functional assignments. Specifically, we showed that the amsJ gene in E. amylovora encodes a pyruvate transferase enzyme that catalyzes the addition of the terminal pyruvate ketal group to the side chain of the amylovoran EPS repeating units. The corresponding gene, wceJ, in P. stewartii turned out to be non-functional due to a few key mutations in the coding sequence. Comparison of the wceJ sequence from several independent natural isolates of P. stewartii revealed identical mutations, thus strengthening our prediction that wceJ became obsolete during adaptation to a different lifestyle and plant host. Creation of some of these mutations in amsJ of E. amylovora led to the production of amylovoran devoid of pyruvate. These findings explain why wild type amylovoran is terminally pyruvylated, while stewartan is terminally glucosylated. We also identified the specific stewartan terminal glucosyl transferase gene in a previously unknown stewartan EPS biosynthetic locus. This same glucosyl transferase gene system exists in E. amylovora, however, which is capable of adding terminal glucosyl residues only when amsJ is deleted. A scenario emerges in which P. stewartii evolved a robust terminal glucosyl transferase accompanied with the loss of the pyruvate transferase activity, while E. amylovora may have benefitted from a highly active terminal pyruvate transferase, and a minimally active of terminal glucosyl transferase. The second non-essential gene, wceF, encodes a stewartan-specific EPS hydrolase, not as reported by other groups, an EPS polymerase. This gene appears to have derived from a phage tailspike protein, which inserted into the stewartan biosynthetic locus to be expressed concomitantly with stewartan EPS synthesis. WceF degrades stewartan EPS gradually beginning in the older part of bacterial colonies, suggesting that this protein is released into the extracellular milieu during cell lysis (WceF is a periplasmic protein). WceF also functions as a chain-length determinant for lipid-linked stewartan capsular polysaccharide (CPS). Crude lipopolysaccharide (LPS) extracts from a wceF mutant strain reveal a high-molecular weight stewartan-specific CPS band during electrophoretic fractionation. Extracts obtained from the wild type strain and the mutant complemented with a functional wceF gene lack this band. We infer that WceF-mediated hydrolysis shortens stewartan CPS, and this band is masked by the bacterial O-antigen fraction. Given the likely phage origin of wceF, we predicted WceF protects P. stewartii from phage infection. In fact, the wceF mutant strain is 10 % more susceptible to phage infection. This level of protection is significant considering that P. stewartii overwinters in the gut of an insect vector where the bacteria are exposed to a variety of bacteriophages. E. amylovora overwinters in the plant host and minimally exposed to phage pressure. Certain phages are reported to be specific for pyruvylated bacterial polysaccharides, perhaps the terminally glucosylated form of stewartan represents an additional protective mechanism. In case of E. amylovora, pyruvylated forms of EPS are generally more "sticky". The bacteria are carried to the blossom of the plant host by insects that pick up the bacterium from the bacterial ooze in spring. The calcium ion sequestration potential of pyruvylated amylovoran may also protect the invading bacteria from calcium-dependent plant innate immune responses. Broader Impacts 1. Research. This study offers important new insights into the potential role of chemically divergent forms of EPS/CPS for bacterial survival in nature and adaptation to specific hosts and thus may provide novel insights and targets for disease control. 2. Education. The award supported the training of 3 undergraduate students, one who is an an author on two published manuscripts. The project supported 3 Ph.D. students (Carlier, Tien, Wang) and two postdoctoral researchers; Dr. Roper is a Professor at the UC-Riverside; Dr. Tsaltas is a Professor at the Cyprus University of Technology, Limassol, Cyprus. 3. Dissemination of Results. The two awards together led to 10 journal publications, plus one manuscript accepted, with two in preparation. Two of the manuscripts are invited reviews. This work was presented at different national and international scientific meetings.
