AbstractA facultative pink-pigmented methylotroph, Methylorubrum populi PINKEL, was isolated from compost containing coffee grounds via a selective enrichment strategy using caffeine (1,3,7-trimethylxanthine) as the sole carbon, nitrogen, and energy source. In addition to caffeine, M. populi PINKEL was also able to grow on other methylxanthines, including theobromine (3,7-dimethylxanthine), paraxanthine (1,7-dimethylxanthine), 7-methylxanthine, and xanthine. Whole cell biodegradation assays in combination with HPLC analysis of spent media confirmed that M. populi PINKEL uses the N-demethylation pathway to degrade caffeine to a mixture of theobromine and paraxanthine. Both of these dimethylxanthines get converted to xanthine via two consecutive N-demethylation steps, with 7-methylxanthine as the intermediate. In silico comparison of the Pseudomonas putida CBB5 ndm genes and the genome of Methylorubrum populi PINKEL helped identify genes that are predicted to encode enzymes of the N-demethylation pathway (amino acid percent identities ranging from 41% to 65%). The ndm genes in M. populi PINKEL are organized in two separate clusters: the first cluster is approximately 3 kb in length and contains the ndmA gene (N1-demethylase; peg_1790); whereas the second cluster measures approximately 11.2 kb and contains the remaining four ndm genes: ndmB (N3-demethylase; peg_1782), ndmC (N7-demethylase; peg_1777), ndmD (reductase; peg_1781), and ndmE (peg_1776). Growth curve and HPLC analysis of each of the five Δndm mutants as well as the corresponding complemented strains helped determine not only which step of the pathway is affected by each individual ndm deletion, but that NdmA is responsible for the N1 and N3 demethylation of caffeine during the first step of the pathway. Transcriptional promoter fusion assays identified the three promoter regions that participate in the transcriptional regulatory system of the five ndm genes, each upstream of the following three genes: ndmA, ndmB, and ndmC. The inability of a ΔndmR (predicted to encode a LysR-type transcriptional regulator) mutant to upregulate expression from any of the three ndm promoter regions during inducing or un-inducing conditions indicates that the LysR protein positively regulates the N-demethylation pathway in M. populi PINKEL. Additionally, it was determined, that any of the intermediates of the N-demethylation pathway containing a methyl group can serve as an inducer of the pathway, although at different levels of induction. This report describes research on the caffeine-degrading bacterium Methylorubrum populi strain PINKEL as a model to study caffeine biodegradation and the characterization of the transcriptional regulatory system for caffeine-degrading enzymes.