Pristinamycin-inducible gene regulation in mycobacteria.
In this work the Pip-inducible system, already used in eukaryotes, was tested in mycobacteria. This system is based on the Streptomyces coelicolor Pip repressor, the Streptomyces pristinaespiralis ptr promoter and the inducer pristinamycin I. By cloning in an integrative plasmid the ptr promoter upstream of the lacZ reporter gene and the pip gene under the control of a constitutive mycobacterial promoter, we demonstrated that the ptr promoter activity increased up to 50-fold in Mycobacterium smegmatis and up to 400-fold in Mycobacterium tuberculosis, in dependence on pristinamycin I concentration, and that the promoter was fully repressed in the absence of the inducer. Three mycobacterial genes were cloned under pptr-Pip control, both in sense and antisense direction; both proteins and antisense RNAs could be over-expressed, the antisenses causing a partial reduction of the amount of the targeted proteins. This system was used to obtain two M. tuberculosis conditional mutants in the fadD32 and pknB genes: the mutant strains grew only in the presence of the inducer pristinamycin I. Thus it showed to be an effective inducible system in mycobacteria.
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26 - 20133 Milano, Italy. francesca.forti@unimi.it
Francesca
F
Forti
Andrea
A
Crosta
Daniela
D
Ghisotti
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects* / Q000235:genetics; D009161:Mycobacterium / Q000187:drug effects* / Q000235:genetics*; D025762:Pristinamycin / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D025762:Pristinamycin