First report of a tetracycline-inducible gene expression system for mollicutes.
Inducible promoter systems are powerful tools for studying gene function in prokaryotes but have never been shown to function in mollicutes. In this study we evaluated the efficacy of the tetracycline-inducible promoter Pxyl/tetO(2) from Bacillus subtilis in controlling gene expression in two mollicutes, the plant pathogen Spiroplasma citri and the animal pathogen Mycoplasma agalactiae. An S. citri plasmid carrying the spiralin gene under the control of the xyl/tetO(2) tetracycline-inducible promoter and the TetR repressor gene under the control of a constitutive spiroplasmal promoter was introduced into the spiralin-less S. citri mutant GII3-9a3. In the absence of tetracycline, expression of TetR almost completely abolished expression of spiralin from the xyl/tetO(2) promoter. Adding tetracycline (>50 ng ml(-1)) to the medium induced high-level expression of spiralin. Interestingly, inducible expression of spiralin was also detected in vivo: in S. citri-infected leafhoppers fed on tetracycline-containing medium and in S. citri-infected plants watered with tetracycline. A similar construct was introduced into the M. agalactiae chromosome through transposition. Tetracycline-induced expression of spiralin proved the TetR-Pxyl/tetO(2) system to be functional in the ruminant pathogen, suggesting that this tetracycline-inducible promoter system might be of general use in mollicutes.
Université de Bordeaux 2, UMR 1090 Génomique Diversité Pouvoir Pathogène, F-33883 Villenave d'Ornon, France.|INRA, UMR 1090 Génomique Diversité et Pouvoir Pathogène, F-33883 Villenave d'Ornon, France.
Marc
M
Breton
Université de Toulouse, ENVT, UMR 1225 Interactions hôtes agents pathogènes, F-31076 Toulouse, France.|INRA, UMR 1225 Interactions hôtes agents pathogènes, F-31076 Toulouse, France.
Evelyne
E
Sagné
Université de Bordeaux 2, UMR 1090 Génomique Diversité Pouvoir Pathogène, F-33883 Villenave d'Ornon, France.|INRA, UMR 1090 Génomique Diversité et Pouvoir Pathogène, F-33883 Villenave d'Ornon, France.
Sybille
S
Duret
Université de Bordeaux 2, UMR 1090 Génomique Diversité Pouvoir Pathogène, F-33883 Villenave d'Ornon, France.|INRA, UMR 1090 Génomique Diversité et Pouvoir Pathogène, F-33883 Villenave d'Ornon, France.
Laure
L
Béven
Université de Toulouse, ENVT, UMR 1225 Interactions hôtes agents pathogènes, F-31076 Toulouse, France.|INRA, UMR 1225 Interactions hôtes agents pathogènes, F-31076 Toulouse, France.
Christine
C
Citti
Université de Bordeaux 2, UMR 1090 Génomique Diversité Pouvoir Pathogène, F-33883 Villenave d'Ornon, France.|INRA, UMR 1090 Génomique Diversité et Pouvoir Pathogène, F-33883 Villenave d'Ornon, France.
Joël
J
Renaudin
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D030008:Catharanthus / Q000382:microbiology; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D006430:Hemiptera / Q000382:microbiology; D045644:Mycoplasma agalactiae / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D012097:Repressor Proteins / Q000235:genetics; D045642:Spiroplasma citri / Q000235:genetics*; D013752:Tetracycline / Q000494:pharmacology*
D001425:Bacterial Outer Membrane Proteins; D012097:Repressor Proteins; C028588:spiralin; C034811:tetracycline resistance-encoding transposon repressor protein; D013752:Tetracycline