Title

Characterization of exopolysaccharide biosynthetic genes -- wceF, wceJ, wzx1, of Pantoea stewartii subsp. stewartii and their corresponding homologues of Erwinia amylovora

Date of Completion

January 2011

Keywords

Biology, Botany|Biology, Genetics|Biology, Microbiology

Degree

Ph.D.

Abstract

Pantoea stewartii subsp. stewartii and Erwinia amylovora are both Gram-negative, xylem dwelling phytopathogens. Cell density-dependent production of capsular/exopolysaccharide (CPS/EPS) is required for the successful colonization within the host's xylem and disease development for both pathogens. The gene clusters encoding the machinery responsible for the biosynthesis of their EPS have been widely studied but the annotations of a few members of the gene clusters heavily rely on bioinformatics predication without any experimental evidence. In this dissertation research, I used genetic and biochemical approaches to validate the function of asmJ and asmL of E. amylovora. They serve as pyruvyl transferase and amylovoran repeating unit flippase, respectively just as predicted. I also showed their corresponding homologues-wceJ and wzx1 does not contribute to the biosynthesis of native stewartan EPS. WceJ is non-functional due to mutation(s) of key amino acid residue(s) while Wzx1 is exclusively responsible for translocating non-native, pyruvylated stewartan repeating units. I also revealed the presence of a high-molecular-weight (HMW), lipid A-linked stewartan and this polymer is subject to WceF-mediated degradation. The degradation of cell-bound HMW stewartan offers a significant level of protection against stewartan-dependent bacteriophage by minimizing the number of surface receptors. ^