Expression and regulation of levansucrase in Pseudomonas syringae

  • Pseudomonas syringae pv. glycinea PG4180 is an opportunistic plant pathogen which causes bacterial blight disease of soybean (Glycine max). This organism can utilize sucrose, the most abundant plant sugar, with the help of enzyme levansucrase (Lsc). Sucrose is eventually cleaved into glucose, which is further metabolised by the cell, and fructosyl residues, which are polymerized by the same enzyme to form the exopolysaccharide levan. PG4180 has three genes for Lsc, of which two, lscB and lscC are expressed while lscA is not expressed. The first part of this study dealt with determining the presence of multiple lsc genes in P. syringae. Nucleotide sequence alignments between lscB/C and lscA helped identify a prophage associated promoter element present in the upstream of lscB/C ORF but not associated with lscA. We could, for the first time, express lscA in PG4180 under the lscB promoter. However, lscB could not be expressed under the lscA promoter indicating that lscA upstream region does not contain a promoter region. We speculate that lscA might be an ancestral Lsc variant in front of which an active promoter, possibly derived from a bacteriophage, got inserted leading to expression of lscB/C. The second part of this study dealt with identifying transcription factors involved in regulation of Lsc expression in P. syringae. The binding site for a hexose metabolism regulator, HexR, was identified in the promoter region of lscB. As compared to the wild-type, the hexR mutant had severely hampered growth characteristics in medium containing glucose or sucrose as sole source of carbon as well as in planta. DNA affinity chromatography was done to identify two H-NS-like regulatory proteins MvaT-MvaU binding to the promoter region of lscB. Protein-DNA binding assay and overexpression of MvaU in PG4180 suggested that these two proteins could act as repressors of lsc expression. The data suggest a co-regulation of genes involved in extra-cellular sugar acquisition with those involved in intra-cellular energy-providing metabolic pathways as well as virulence and biofilm expression in P. syringae.

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Publishing Institution:IRC-Library, Information Resource Center der Jacobs University Bremen
Granting Institution:Jacobs Univ.
Author:Shaunak Khandekar
Referee:Matthias Ullrich, Reimer Stick, Georgi Muskhelishvili
Advisor:Matthias Ullrich
Persistent Identifier (URN):urn:nbn:de:101:1-201307119491
Document Type:PhD Thesis
Date of Successful Oral Defense:2013/04/16
Date of First Publication:2013/04/30
PhD Degree:Biochemistry
School:SES School of Engineering and Science
Library of Congress Classification:Q Science / QR Microbiology / QR75-99.5 Bacteria
Call No:Thesis 2013/6

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