The Escherichia coli rhamnose promoter rhaP(BAD) is in Pseudomonas putida KT2440 independent of Crp-cAMP activation.
We developed an expression vector system based on the broad host range plasmid pBBR1MCS2 with the Escherichia coli rhamnose-inducible expression system for applications in Pseudomonas. For validation and comparison to E. coli, enhanced green fluorescent protein (eGFP) was used as a reporter. For further characterization, we also constructed plasmids containing different modifications of the rhaP(BAD) promoter. Induction experiments after the successful transfer of these plasmids into Pseudomonas putida KT2440 wild-type and different knockout strains revealed significant differences. In Pseudomonas, we observed no catabolite repression of the rhaP(BAD) promoter, and in contrast to E. coli, the binding of cyclic adenosine monophosphate (cAMP) receptor protein (Crp)-cAMP to this promoter is not necessary for induction as shown by deletion of the Crp binding site. The crp(-) mutant of P. putida KT2440 lacked eGFP expression, but this is likely due to problems in rhamnose uptake, since this defect was complemented by the insertion of the L-rhamnose-specific transporter rhaT into its genome via transposon mutagenesis. Other global regulators like Crc, PtsN, and CyoB had no or minor effects on rhamnose-induced eGFP expression. Therefore, this expression system may also be generally useful for Pseudomonas and other gamma-proteobacteria.
Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
Marcel
M
Jeske
Josef
J
Altenbuchner
D000242:Cyclic AMP / Q000235:genetics / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein / Q000235:genetics / Q000378:metabolism*; D004251:DNA Transposable Elements / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism*; D012210:Rhamnose / Q000235:genetics* / Q000378:metabolism*
D002373:Cyclic AMP Receptor Protein; D004251:DNA Transposable Elements; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; D012210:Rhamnose