1.6.5
CHARACTERIZATION OF A MORPHINONE REDUCTASE HOMOLOGUE IN PSEUDOMONAS SYRINGAE PV. GLYCINEA

B ROHDE1, R SCHMID2 and M ULLRICH1

1Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, Germany; 2FB5-Mikrobiologie, Universität Osnabrück, Germany

Background and objectives
Temperature as an environmental signal plays an important role during the host-parasite interaction. Bacterial blight, the disease caused by P. syringae on several host plants, mainly occurs under cold weather conditions. The phytopathogen might sense temperature changes which lead to the transition from epiphytic growth to the virulent phenotype. To investigate the temperature-dependent expression of virulence factors in the P. syringae pv. glycinea PG4180.N9 cells were grown at 28°C (optimal growth temperature) and at 18°C (optimal temperature for the production of the phytotoxin coronatine) [1].

Results and conclusions
Cytoplasmic proteins derived from PG4180.N9 cultures at either growth temperature were separated by 2-D gel electrophoresis. Several protein spots which appeared to be more or solely induced at 18°C were N-terminally sequenced. An approximately 40-kDa protein with an isoelectric point of 5.2 revealed significant sequence homology to morphinone reductase (MorB) of P. putida M10 [2]. A 600-bp PCR fragment, amplified with primers derived from the morB sequence of P. putida, was digoxigenin-labelled and used to screen a genomic cosmid library of P. syringae pv. glycinea. A 4.3-kb EcoRI-fragment which hybridized under high stringency conditions was subcloned and the morB-homologue of P. syringae was sequenced. Nucleotide sequence comparison of both genes revealed 52% similarity and 48% identity to each other.

A PCR screening with different Pseudomonas strains was carried out using primers generated from the morB sequence of Pseudomonas syringae pv. glycinea. To confirm positive strains, the PCR products were blotted on nitrocellulose membranes and subjected to Southern blot analysis. It revealed that the morB gene is widespread among P. syringae pv. glycinea strains, but not in other pathovars of P. syringae. The morB gene was cloned into pMal-C2 and overexpressed in E. coli cells. The MBP-MorB fusion protein was purified by affinity chromatography on amylose resin. The purified fusion protein was then cleaved by Factor Xa protease, separated by SDS-PAGE, and the 40-kDa MorB protein was used to immunize rabbits. So far, a substrate for the MorB-homologue could not be identified. As shown for morphinone reductase of P. putida, enzymatic activity was measured with morphinone, codeinone, neopinone and 2-cyclohexen-1-one as substrates [2]. Morphinone belongs to the alkaloids. Several alkaloids and phytoalexins are produced by plants and possess antimicrobial activity. P. syringae might have developed a defence mechanism, in which MorB plays an important role in the detoxification of antimicrobial agents. Other possible groups of compounds which might act as substrates are structurally related phytoalexins, chalcones and flavones. The enzymatic characterization is currently investigated.

References
1. Budde IP, Rohde BH, Bender CL, Ullrich M, 1998. Journal of Bacteriology (in press).
2. French CE, Bruce NC, 1994. Biochemical Journal 301, 97-103.