(175ac) Characterization of the Transcription Regulators Cafr and Caft from Pseudomonas Putida CBB5

The soil bacterium Pseudomonas putida CBB5, first isolated in 2009, is able to metabolize caffeine, theophylline, and related methylxanthines as sole carbon and nitrogen sources¹. This process in CBB5 is the result of a five component Rieske oxygenase N-demethylation system, composed of the enzymes NdmABCDE which sequentially N-demethylate caffeine to xanthine. These enzymes have been extensively researched since their discovery, and we are now focusing on how the enzymes are regulated in the cells. There are two known alkylxanthine-responsive transcriptional regulatory proteins that govern NdmABCDE in CBB5². These regulatory proteins, CafR and CafT, are induced by caffeine³. CafR and CafT bind to promoters located near NdmA and NdmB to control the gene expression³.

In this project, we have partially characterized the CafR and CafT proteins in P. putida CBB5. Single knockout studies indicate that CafR is a repressor protein that blocks the expression of the ndmABCDE genes in the absence of caffeine, while CafT is an inducer that increases gene expression in the presence of caffeine. We have further determined the effect of caffeine and theophylline on ndmABCDE gene expression in the knockout strains and are working to determine the minimal promoter sequences for the N-demethylase genes.

1. Yu, C. L.; Louie, T. M.; Summers, R.; Kale, Y.; Gopishetty, S.; Subramanian, M., Two Distinct Pathways for Metabolism of Theophylline and Caffeine Are Coexpressed in Pseudomonas putida CBB5. Journal of Bacteriology 2009, 191 (14), 4624-4632.
2. Quandt, E. M.; Summers, R. M.; Subramanian, M. V.; Barrick, J. E., Draft Genome Sequence of the Bacterium Pseudomonas putida CBB5, Which Can Utilize Caffeine as a Sole Carbon and Nitrogen Source. Genome Announc 2015, 3 (3), e00640-15.
3. Venkiteswaran Subramanian, R. S., Michael Tai-Man Louie Caffeine responsive promoters and regulatory genes. 2013.