title: One-plasmid tunable coexpression for mycobacterial protein-protein interaction studies.
abstract: A single plasmid that allows controlled coexpression has been developed for use in mycobacteria. The tetracycline inducible promoter, PtetO, was used to provide tetracycline-dependent induction of one gene, while the Psmyc, Pimyc, or Phsp promoters were used to provide three different levels of constitutive expression of a second gene. The functions of these four individual promoters were established using green fluorescent protein (GFP) and a newly identified red fluorescence inducible protein from Geobacillus sterothermophilus strain G1.13 (RFIP) as reporters. The tandem use of GFP and RFIP as reporter genes allowed optimization of the tunable coexpression in Mycobacterium smegmatis; either time at a fixed inducer concentration or changes in inducer concentration could be used to control the protein:protein ratio. This single vector system was used to coexpress the two-protein Mycobacterium tuberculosis stearoyl-CoA Delta(9) desaturase complex (integral membrane desaturase Rv3229c and NADPH oxidoreductase Rv3230c) in M. smegmatis. The catalytic activity was found to increase in a manner corresponding to increasing the level of Rv3230c relative to a fixed level of Rv3229c. This system, which can yield finely tuned coexpression of the fatty acid desaturase complex in mycobacteria, may be useful for study of other multicomponent complexes. Furthermore, the tunable coexpression strategy used herein should also be applicable in other species with minor modifications.
authors.affiliation: Biophysics Graduate Degree Program, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706-1549, USA.
authors.forename: Yong
authors.initials: Y
authors.lastname: Chang
authors.forename: David
authors.initials: D
authors.lastname: Mead
authors.forename: Vinay
authors.initials: V
authors.lastname: Dhodda
authors.forename: Phil
authors.initials: P
authors.lastname: Brumm
authors.forename: Brian G
authors.initials: BG
authors.lastname: Fox
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002452:Cell Count; D003001:Cloning, Molecular / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D025941:Protein Interaction Mapping / Q000379:methods*; D013230:Stearoyl-CoA Desaturase / Q000235:genetics / Q000378:metabolism; D013752:Tetracycline; D000097573:Red Fluorescent Protein
chemical_list: D001426:Bacterial Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D013230:Stearoyl-CoA Desaturase; D013752:Tetracycline; D000097573:Red Fluorescent Protein