1.5.2
EFFECTS OF PTS1 MUTATIONS ON ENZYME SYNTHESIS AND VIRULENCE IN ERWINIA CHRYSANTHEMI

A EVTUSHENKOV, K DATSENKO and Y FOMICHEV

Department of Microbiology, Belarus State University, Minsk, Belarus

Background and objectives
The phytopathogenicity of pectolytic bacteria of the genus Erwinia is due to their capacity to produce depolymerizing enzymes, which degrade the components of plant cell walls. Pectate lyases play a major role in such processes. Pectinase synthesis is regulated by different extracellular and intracellular factors, one of which is catabolite repression. The phosphotransferase system (PTS) of bacteria plays a key role in mechanisms of catabolite repression. This work aims to elucidate the role of the pts1 gene of PTS in enzyme synthesis and pathogenicity of Erwinia species.

Materials and methods
The strains of E. chrysanthemi ENA49 and E. carotovora subsp. atroseptica 3-2 were mutagenized by transposon Tn5. pts1 genes were cloned on the plasmid pULB113. Pectate lyase analysis was performed by electrophoresis in polyacrylamide gel. Virulence was tested on potato tuber slices and potato stems.

Results and conclusions
E. chrysanthemi pts1 and E. carotovora subsp. atroseptica pts1 mutants did not ferment glucose, fructose, mannitol or mannose on indicator plates. They slowly fermented glycerol, arabinose and galactose. The mutant bacteria had altered transport and phosphorylation of alpha-methylglucoside, glucose, fructose, mannitol and mannose. The mutant phenotype was complemented by the pULB113 plasmid with cloned pts1 genes of E. chrysanthemi ENA49 and E. carotovora subsp. atroseptica 3-2. Nucleotide sequencing of the DNA fragment adjacent to the site of Tn5 insertion revealed high similarity to the pts1 gene of Escherichia coli.

E. chrysanthemi pts1 F'lac had a ca twofold decrease in the production of beta-galactosidase and pectate lyase. The synthesis of these two enzymes in E. chrysanthemi pts1 F'lac strain was not repressed by glucose, whereas wild-type bacteria had 80-90% repression. Although the total pectate lyase activity was lower in the pts1 mutant, the production of pectate lyases b and c was increased. The mutant strain also had a more pronounced decrease of pectate lyase activity when grown at high temperature. At the same time, no significant difference was observed in the rate of maceration of the potato tuber tissue by the mutant and wild-type strains both at 28 and 37C.

E. carotovora subsp. atroseptica pts1 mutant had a twofold decrease in the production of beta-galactosidase and a ca 10-fold decrease in pectate lyase activity. Neither pectate lyase nor beta-galactosidase was repressed by glucose in the E. carotovora subsp. atroseptica pts1 strain. The mutant bacteria were not able to cause blackleg symptoms and had reduced ability to macerate potato tuber slices.