Identification and characterization of transcriptional regulators responsible for the temperature-dependent expression of Pseudomonas syringae levansucrase gene
Temperature-dependent expression of virulence factors, such as exopolysaccharides, is crucial for many pathogenic bacteria. In the plant pathogenic bacterium, Pseudomonas syringae pv. glycinea PG4180, synthesis of the exopolysaccharide levan is mediated by 2 levansucrases, encoded by lscB and lscC, which are expressed in a temperature-dependent manner: maximum mRNA production for these genes occurs at 18°, when the pathogen is highly virulent, and is repressed at 28°, the bacterium’s optimal growth temperature. By means of DNA affinity chromatography and subsequent MALDI-Tof analysis we have identified 3 repressors, which are responsible for silencing lscB gene at 28°, but do not bind the lscB promoter at 18°. In order to find which factors might determine this temperature-dependent binding of repressors, a screening of P. syringae genomic library for the lscB expression activator in the heterologous host, P. putida, was performed. P. putida does not have any levansucrase genes in its genome. Moreover, the lscB gene from P. syringae was not expressed under control of its native in P. putida at any temperature. When the genomic library of P. syringae was conjugated into P. putida (lscB+), three cosmids were found to promote lscB expression at 18°, but not at 28°. A single gene designated lscR, was common to all three cosmids and its presence was sufficient to induce lscB expression.
Overall, our data suggested that expression of lscB is dependent on reversible binding of at least three repressors to its upstream regulatory sequence, and this reversible binding might in turn be regulated by the activator protein, LscR. Mutants creation and overexpression of the identified transcriptional regulators are underway.
This project is supported by Marie Curie EST Grant (MarMic program, Max Planck Institute Bremen)
The project is run in collaboration with the groups of Prof. B. Eikmanns (University Ulm) and Prof. M.Bott (Forschungszentrum Julich)
Fig. 1 Identification of one of the repressor protein, bound to IscB promoter at 28, but not 18.
Fig. 2 Screening of genomic library from P. syringae in P.putida (IscB+) the arrow indicates levan-producing clone