1.5.8
CHARACTERIZATION OF THE ERWINIA AMYLOVORA rpoS GENE

JA EASTGATE, M ANDERSON and IS ROBERTS

School of Biological Sciences, University of Manchester, Oxford Road, Manchester, UK

Background and objectives
Erwinia amylovora is the causative organism of the disease fireblight, which affects members of the Pomoideae (apples and pears) and ornamental plant species. The pathogenesis of fireblight infection has been well studied at a cellular level and three major pathogenicity determinants identified: the surface exopolysaccharide, the harpin biosynthetic/secretory system, and the products of the dsp gene cluster [1]. It has been proposed that on entering the host environment nutrient limitation is experienced by a pathogen, leading to expression of genes required for in vivo survival and virulence. As such, expression of the alternative sigma factor RpoS, required to maintain viability during periods of starvation and environmental stress [2], is required for the pathogenicity of certain mammalian pathogens. In an attempt to understand the mechanism of the host-bacterial interactions occurring during the fireblight disease process, we cloned and characterized the E. amylovora rpoS gene.

Results and conclusions
The gene encoding the alternative sigma factor RpoS was cloned from E. amylovora by complementation of an E. coli rpoS mutant strain with a reporter fusion in the bolA gene. The nucleotide sequence of E. amylovora rpoS was determined and found to be 81% homologous to the E. coli gene. The E. amylovora rpoS gene was found to be situated 3 of an nlpD homologue and 5 of a mutS homologue, a similar organization to that found in E. coli. The predicted amino acid sequence was 97% homologous to RpoS of E. coli and Salmonella species, and 96% homologous to E. carotovora and Shigella flexneri, and the protein was recognized by antibodies generated against E. coli RpoS. Transcript mapping of the rpoS gene identified a single start site 536 bases 5 to rpoS, located within the coding sequence of the nlpD gene. The chromosomal copy of E. amylovora rpoS was insertionally inactivated, the resulting rpoS mutant showed reduced ability to survive in stationary-phase culture and increased sensitivity to oxidative and osmotic stresses. However, when assayed for pathogenicity the mutant was not affected in its ability to cause disease symptoms in the apple seedling model.

Nutrient limitation has been suggested to be an important signal during disease processes, inducing expression of virulence determinants and metabolic functions essential for bacterial survival within the host environment. In addition to its role in stress survival, functional RpoS has been shown be important for the virulence of a number of animal pathogens The E. amylovora rpoS mutant showed increased sensitivity to a number of environment stresses but was not compromised in its ability to cause disease. This suggests the absence of a direct role for rpoS in the pathogenic process. However, it is unclear how nutritionally limited plant tissue is to a pathogen; the level of glucose in the apoplast has been estimated at 3-6 mM, which is in excess of the 10-4 mM glucose concentration thought to induce the onset of stationary phase. In addition, localized release of nutrients as a result of trauma at the inoculation site may mask starvation stimulus in our assay.

References
1. Roberts IS, Coleman MJ, 1991. Journal of General Microbiology 137, 1453-1457.
2. Kolter R, Siegele DA, Tormo A, 1993. Annual Review of Microbiology 47, 855-874.