Regulation of the Levansucrase Genes from Pseudomonas syringae pv. glycinea at the Level of Transcription
In the plant pathogenic bacterium Pseudomonas syringae pv. glycinea PG4180 the exopolysaccharide levan is synthesized by two levansucrases, LscB and LscC. Their respective genes, lscB and lscC, are expressed temperature- dependently, with maximum transcription at 18°C. Temperature-responsive expression relies on a particular promoter structure of the levansucrase genes as well as on regulators, which mediate this process. Structure of the lscB upstream sequence was analyzed by nested deletion analysis in order to map the minimal promoter sequence and presumable thermo- and substrate sensory regions. Current results suggest that at least roughly 1 kb upstream of lscB is necessary to express levansucrase. It was also shown that not all P. syringae strains exhibit the same thermo-responsive synthesis of the levansucrases as PG4180. Comparison of the levansucrases promoter sequences from different Pseudomonas strains to identify the regulator-binding sites is currently in progress. In order to reveal trans-acting regulators, which are responsible for levansucrases expression, lscB and lscC genes together with their presumable native promoters were brought separately into the heterologous host Pseudomonas putida KT2440 strain. Neither lscC nor lscB induced levan formation in P. putida. However, three cosmids from a PG4180 genomic library were found to induce levan formation in P. putida (lscC+) and P. putida (lscB+). Restriction pattern of these cosmids revealed several common fragments, which together comprise approximately 12 kb. Transposon in vitro mutagenesis was performed for the identification of the particular levansucrase gene(s), responsible for levansucrase gene expression. Nucleotide sequencing of the region containing the potential regulator is underway.
KeywordsExopolysaccharide nested deletion analysis heterologous expression
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