Differential green fluorescent protein expression from mycobacterial promoter constructs in Escherichia coli and Mycobacterium marinum.
The Mycobacterium marinum G13 promoter is a sigma 70-like promoter that is more active by green fluorescent protein (gfp) differential fluorescence induction (DFI) assays when M. marium resides in an intracellular compartment as compared with growth in media. In assays using DFI, we found that the mycobacterial G13 promoter was also more active in a background of lower nutrient availability during logarithmic growth. This promoter, contained in an insert cloned upstream of a gfp reporter gene, is also active in Escherichia coli. When gfp expression assays of different plasmid constructs were performed in parallel with E. coli and M. marinum, expression in E. coli was maintained after deletion of both upstream and/or downstream regions proximal to the core promoter sequence. In M. marinum, however, although upstream deletions had no appreciable effect on gfp expression, promoter constructs with deleted downstream regions expressed 20- to 40-fold less gfp over all growth phases. The high-level expression of gfp was restored, however, in a clone containing approximately 100 bp downstream of the transcriptional start point. We have therefore utilized this gfp reporter assay of promoter activity to distinguish possible differences in requirements for gfp expression between different genera that utilize sigma 70-like promoter elements. We found that high levels of expression of gfp from the G13 promoter in M. marinum require downstream regions not necessary for gfp expression in E. coli.
Gall
Kelly
K
Department of Anatomy, Microbiology and Pathology, University of Minnesota Medical School, Duluth, MN 55812, USA.
Barker
Lucia P
LP
D003001:Cloning, Molecular; D003470:Culture Media; D012321:DNA-Directed RNA Polymerases; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D005434:Flow Cytometry; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D009161:Mycobacterium / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012808:Sigma Factor; D014169:Transformation, Bacterial
D003470:Culture Media; D012808:Sigma Factor; D049452:Green Fluorescent Proteins; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases