This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chiribau, C. B. Articles by Brandsch, R. Search for Related Content PubMed PubMed Citation Articles by Chiribau, C. B. Articles by Brandsch, R.

Characterization of PmfR, the Transcriptional Activator of the pAO1-Borne purU-mabO-folD Operon of Arthrobacter nicotinovorans

Calin B. Chiribau,¹ Cristinel Sandu,^1, Gabor L. Igloi,² and Roderich Brandsch^1*

Institute of Biochemistry and Molecular Biology,¹ Institute of Biology III, University of Freiburg, Freiburg, Germany²

Received 15 December 2004/ Accepted 17 January 2005

Nicotine catabolism by Arthrobacter nicotinovorans is linked to the presence of the megaplasmid pAO1. Genes involved in this catabolic pathway are arranged on the plasmid into gene modules according to function. During nicotine degradation -N-methylaminobutyrate is formed from the pyrrolidine ring of nicotine. Analysis of the pAO1 open reading frames (ORF) resulted in identification of the gene encoding a demethylating -N-methylaminobutyrate oxidase (mabO). This gene was shown to form an operon with purU- and folD-like genes. Only in bacteria grown in the presence of nicotine could transcripts of the purU-mabO-folD operon be detected, demonstrating that this operon constitutes part of the pAO1 nicotine regulon. Its transcriptional start site was determined by primer extension analysis. Transcription of the operon was shown to be controlled by a new transcriptional regulator, PmfR, the product of a gene that is transcribed divergently from the purU, mabO, and folD genes. PmfR was purified, and electromobility shift assays and DNase I-nuclease digestion experiments were used to determine that its DNA binding site is located between –48 and –88 nucleotides upstream of the transcriptional start site of the operon. Disruption of pmfR by homologous recombination with a chloramphenicol resistance cassette demonstrated that PmfR acts in vivo as a transcriptional activator. Mutagenesis of the PmfR target DNA suggested that the sequence GTTT-14 bp-AAAC is the core binding site of the regulator upstream of the –35 promoter region of the purU-mabO-folD operon.

Present address: Molecular Biophysics, Rockefeller University, New York, N.Y.

This article has been cited by other articles:

• Ganas, P., Mihasan, M., Igloi, G. L., Brandsch, R. (2007). A two-component small multidrug resistance pump functions as a metabolic valve during nicotine catabolism by Arthrobacter nicotinovorans. Microbiology 153: 1546-1555 [Abstract] [Full Text]  
• Mihasan, M., Chiribau, C.-B., Friedrich, T., Artenie, V., Brandsch, R. (2007). An NAD(P)H-Nicotine Blue Oxidoreductase Is Part of the Nicotine Regulon and May Protect Arthrobacter nicotinovorans from Oxidative Stress during Nicotine Catabolism. Appl. Environ. Microbiol. 73: 2479-2485 [Abstract] [Full Text]  
• Sachelaru, P., Schiltz, E., Brandsch, R. (2006). A Functional mobA Gene for Molybdopterin Cytosine Dinucleotide Cofactor Biosynthesis Is Required for Activity and Holoenzyme Assembly of the Heterotrimeric Nicotine Dehydrogenases of Arthrobacter nicotinovorans.. Appl. Environ. Microbiol. 72: 5126-5131 [Abstract] [Full Text]  
• Zhang, X.-S., Cheng, H.-P. (2006). Identification of Sinorhizobium meliloti Early Symbiotic Genes by Use of a Positive Functional Screen. Appl. Environ. Microbiol. 72: 2738-2748 [Abstract] [Full Text]