1.5.12
BIOSYNTHESIS OF EXOPOLYSACCHARIDES BY THE FIREBLIGHT PATHOGEN ERWINIA AMYLOVORA AND ITS REGULATION

J BOGS, C LANGLOTZ, H ULLRICH, Y ZHANG and K GEIDER

Max-Planck-Institut fur Zellbiologie, Rosenhof, D-69526 Ladenburg/Heidelberg, Germany

Background and objectives
For protection against environmental stress and to reduce recognition by plant defence mechanisms, Erwinia amylovora, which causes fireblight on apple and pear fruit trees, produces exopolysaccharide (EPS), either the homopolymer levan from sucrose in the environment [1] or the complex acidic EPS amylovoran [2]. Synthesis of amylovoran depends on regulatory steps based on two-component systems including the proteins RcsA, RcsB [3] and possibly RcsC. Its synthesis is further affected by environmental conditions, including the carbon source such as sorbitol [4], the pH value of the medium, and the temperature of cultivation.

Results and conclusions
The regulation of the ams-operon by Rcs-proteins, the Ion-protease and by environmental conditions was assayed with amsG::lacZ fusions. Expression of the levansucrase gene does not depend on Rcs-proteins, but seems to be dependent on other regulators, which have been characterized on the molecular level for the rcsA-gene.

The biosynthesis of amylovoran was examined in EDTA-treated cells for incorporation of labelled UDP-galactose. The 'repeating units' were analysed by HPLC, The molecular weight of EPS was determined by SEC-DRI/LALLS. Chain length depended on temperature, carbon source in the media and the genetic background of the strains. An EPS negative mutant of E. amylovora which was complemented with corresponding genes of Erwinia stewartii showed an altered repeating unit of EPS consisting of a stewartan-like backbone and an amylovoran-like side chain. The function of amsM, which has high homology to the galU and galF genes, was studied by genetic and biochemical assays. Chromosomal amsM mutants could not grow on galactose and were EPS negative. Unexpectedly, the GalU activity of these mutants was higher than in the parental strain and reduced after overexpression of amsM. Genes encoding extracytoplasmic proteins were identified by TnMax mutagenesis. The cellular localization of AmsH, AmsF and levansucrase could thus be determined.

Host plants for fireblight contain sorbitol and sucrose as storage and transport sugars. Sorbitol and sucrose mutants are strongly reduced in virulence against the host plants. Their molecular genetics were characterized for the cell metabolism of E. amylovora. The green fluorescent protein (GFP) was used as a reporter for promoter activity of the corresponding operons analysed with a fluorometer and FACS (fluorescent activated cell scanner). GFP was also applied to follow the movement of E. amylovora in the intact tissue of host plants. The pathogen moved against the water stream in the xylem, clogged the vessels and caused outbreaks into the parenchyma. An EPS-negative mutant was not able to migrate or multiply in plant tissue. Inoculation into the intercostal region of leaves or on the leaf surface resulted in migration into the vascular bundles.

Synthesis of the capsular EPS amylovoran is strictly required for pathogenicity of E. amylovora. The metabolism of carbon sources was genetically determined and regulation of the ams-operon measured with fusions of the reporter gene and ams-promoter fragments to follow the movement of E. amylovora in plant tissue, including sensing of induction of amylovoran synthesis. The chain length of amylovoran is relatively stable and depends on transport proteins. Interference with these functions may help to control capsule bacterial formation, and its reduction should expose E. amylovora to plant defence mechanisms.

References
1. Geier G, Geider K, 1993. Physiological and Molecular Plant Pathology 42, 387-404.
2. Bugert P, Geider K, 1995. Molecular Microbiology 15, 917-933.
3. Bereswill S, Geider K, 1997. Journal of Bacteriology 179, 1354-1361.
4. Aldridge P, Metzger M, Geider K, 1997. Molecular and General Genetics 256, 611-619.