Isolation of novel Pseudomonas syringae promoters and functional characterization in polyhydroxyalkanoate-producing pseudomonads.

A library of genomic DNA fragments of Pseudomonas syringae pv. tomato DC3000 was constructed in a lacZalpha-containing plasmid, pBS29. The library was used in a preliminary alpha-complementation-based screen to identify clones with promoter activity in Escherichia coli. Ten positive clones were sequenced and their locations in the chromosomal DNA of DC3000 strain were mapped. Five positive clones (P2, P3, P4, P6 and P8) were further assayed for promoter activity in three polyhydroxyalkanoate-producing pseudomonads: Pseudomonas resinovorans, P. corrugata and P. chlororaphis. To this end, a green-fluorescent-protein gene (gfp) was cloned downstream from the putative (DC3000) promoter in a shuttle plasmid. We found that only Pseudomonas transformants harboring the gfp-containing plasmid driven by putative promoter P2 showed fluorescence, indicating that this promoter is functioning in the three tested pseudomonads. Results of an in silico analysis of the P2 sequence further support the assignment of P2 as a bona fide promoter by the localization of putative -10 and -35 promoter regions and a transcription-factor-binding site, rpoD17, in this sequence. We successfully applied promoter P2 to drive the expression in P. chlororaphis of a recombinant alpha-galactosidase gene of Streptomyces coelicolor, which should be useful for the utilization of oligosaccharides of soy molasses for the production of polyhydroxyalkanoate biopolymer or rhamnolipid biosurfactant.