Development of an efficient expression system for Flavobacterium strains

Strong promoters were isolated from Flavobacterium johnsoniae in a promoter-trap vector incorporating a gfp reporter system, and were used to express fluorescent protein markers (including GFP, YFP, mOrange and mStrawberry) and insecticidal protein genes in Flavobacterium strains. Sequence analysis of trapped DNA fragments showed conserved Bacteroidetes promoter motifs (TTG-N19-TAnnTTTG) located upstream of putative open reading frames. Plasmids harboring these genomic DNA fragments from F. johnsoniae promoted strong production of fluorescent proteins in Flavobacterium hibernum but not in Escherichiacoli. The most potent promoter (PompA) identified in this work was cloned upstream of genes encoding fluorescent proteins, and these were co-expressed in Flavobacterium strains. The p42 and p51 genes (binary toxins from Bacillus sphaericus) when translationally fused to the 3′-end of gfp showed strong expression. Flavobacteria expressing these genes exhibited toxicity against larvae of the mosquitoes Culex quinquefasciatus, Anopheles gambiae, and Ochlerotatus triseriatus. However, transformants with the transcriptional fusion construct between cry11A with p20 from Bacillus thuringiensis did not express Cry11A protein indicating that constitutive expression of cry11A may be problematic in Flavobacterium.