1.9.1
MUTANTS OF ERWINIA CAROTOVORA SUBSP. ATROSEPTICA WITH ALTERED RESISTANCE TO PLANT PHENOLIC COMPOUNDS

AG PESHYAKEVICH, YA NIKOLAICHIK and TM ANKUDO

Department of Microbiology, Byelorussian State University, Minsk 220050, Belarus

Background and objectives
Although the antimicrobial activity of phenolics against Erwinia has been demonstrated [1], no information is available on special genetic systems of Erwinia controlling resistance to plant phenolic compounds. The aim of this work was the isolation and characterization of mutants of Erwinia carotovora subsp. atroseptica strain 3-2 (Eca 3-2) with altered resistance to plant phenolics.

Materials and methods
Ferulic and caffeic acids (Koch-Light Laboratories), cinnamic acid (British Drug House), chlorogenic, vanillic and salicylic acids (Sigma) were dissolved in ethanol and these solutions were added to nutrient L-agar ( Sigma) in suitable concentrations. Bacteria were grown in L-broth (Sigma) or L-agar supplemented with 50 g/ml rifampicin, 100 g/ml ampicillin, 50 g/ml kamamycin, 25 g/ml chloramphenicol, 10 g/ml trimethoprim or 50 g/ml streptomycin where appropriate. E.coli was grown at 37C and Erwinia at 28C. Eca3-2 insertion mutants were obtained by mating with E. coli strain S17-1 (lambda pir) pUT/mini-Tn5xylE [2]. To observe catechol-2,3-dioxygenase activity, bacteria were grown on agar and colonies sprayed with 0.5 M catechol. Catechol-2,3-dioxygenase activity was quantified spectrophotometrically after addition of sonicated cells to substrate containing 20 l of 0.1 M catechol in 3 ml phosphate buffer pH 8.0. Pathogenicity of bacteria was tested on soil-grown and micropropagated potato plants cvs Orbita and Yavor.

Results and conclusions
Eca 3-2 mutants with mini-Tn5xylE insertions were screened for the expression of the reporter gene xylE in the presence of 10 g/ml of each phenolic compound. Eca 3-2 derivatives P5162, P5110 and P5141 were selected for further characterization. These mutants appeared to have increased xylE gene expression in response to vanillic acid (P5162, 2.8-fold increase) and salicylic acid (P5110, 7.2-fold increase and P5141, 5-fold increase).

We have shown that the wild-type strain is able to degrade both vanillic and salicylic acids. All three mutants have lost this ability. The mutant strains also have reduced resistance to these phenolic compounds as compared to the wild-type strain. The mutants cannot cause blackleg symptoms in potato plants, but have retained the ability to cause the hypersensitivity reaction in bean leaves. As the production of the main pathogenicity factors (pectinolytic, proteolytic and cellulolytic enzymes) is not altered in the mutant strains, we conclude that genes controlling the resistance to vanillic and salicylic acids play an important role in the determination of the virulence of this blackleg pathogen.

References
1. Lyon GD, McGill FM, 1988. Potato Research 31, 461-467.
2. de Lorenzo V, Herrero M, Jakubzik U, Timmis K, 1990. Journal of Bacteriology 172, 6568-6572.