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Title: Isolation of novel Pseudomonas syringae promoters and functional characterization in polyhydroxyalkanoate-producing pseudomads

Author
item Solaiman, Daniel
item Swingle, Bryan

Submitted to: New Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/9/2009
Publication Date: 2/1/2010
Citation: Solaiman, D., Swingle, B.M. 2010. Isolation of novel Pseudomonas syringae promoters and functional characterization in polyhydroxyalkanoate-producing pseudomads. New Biotechnology. 27(1):1-9.

Interpretive Summary: Pseudomonads are a family of bacteria with agricultural, health and environmental importance. Many pseudomonads are also valuable microbes with a potential use for the production of industrial chemicals such as polyhydroxyalkanoate (a biodegradable plastic) and rhamnolipid (a bio-based surfactant) from agricultural (co)products. Genetic engineering of pseudomonads through the expression of a new gene(s) is a powerful means to manipulate these bacteria to a greater extent. Promoter is a piece of DNA that is needed to express a gene (make it work). It is a common practice to use promoters from E. coli to express new genes in pseudomonads. In so doing, however, other useful characteristics of an indigenous promoter such as promoter strength or regulation may be lost. We initiated a study to find new promoters from a strain of pseudomonad called P. syringae DC3000. Four putative promoters obtained from a preliminary screen in E. coli were then tested in three bacterial strains capable of producing polyhydroxyalkanoate bioplastics. We found that one of these promoters, called P2, is capable of directing the expression of a foreign gene that codes for a green fluorescent protein. Computer analysis of the P2 sequence further supports the identity of this DNA piece as a bona fide promoter. P2 promoter should thus be useful for expressing metabolic gene(s) in pseudomonads to improve their performance in an intended application.

Technical Abstract: A library of genomic DNA fragments of Pseudomonas syringae pv. tomato DC3000 was constructed in a lacZa-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. Four positive clones (P2, P3, P4, and P6) 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 P. syringae 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 the -10 and -35 promoter regions and a transcription factor-binding site for an integration host factor (Ihf) in this sequence.