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Microbiology 153 (2007), 1546-1555; DOI  10.1099/mic.0.2006/004234-0
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Microbiology 153 (2007), 1546-1555; DOI  10.1099/mic.0.2006/004234-0
© 2007 Society for General Microbiology

A two-component small multidrug resistance pump functions as a metabolic valve during nicotine catabolism by Arthrobacter nicotinovorans

Petra Ganas1, Marius Mihasan1,3, Gabor L. Igloi2 and Roderich Brandsch1

1 Institute of Biochemistry and Molecular Biology, Centre for Biochemistry and Molecular Cell Research, Albrecht-Ludwigs University, Freiburg, Germany
2 Institute of Biology III, Albrecht-Ludwigs University, Freiburg, Germany
3 Department of Biochemistry, Alexandru-Ioan-Cuza University, Iasi, Romania

Correspondence
Roderich Brandsch
roderich.brandsch{at}biochemie.uni-freiburg.de

The genes nepAB of a small multidrug resistance (SMR) pump were identified as part of the pAO1-encoded nicotine regulon responsible for nicotine catabolism in Arthrobacter nicotinovorans. When [14C]nicotine was added to the growth medium the bacteria exported the 14C-labelled end product of nicotine catabolism, methylamine. In the presence of the proton-motive force inhibitors 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenylhydrazone (CCCP) or the proton ionophore nigericin, export of methylamine was inhibited and radioactivity accumulated inside the bacteria. Efflux of [14C]nicotine-derived radioactivity from bacteria was also inhibited in a pmfR : cmx strain with downregulated nepAB expression. Because of low amine oxidase levels in the pmfR : cmx strain, {gamma}-N-methylaminobutyrate, the methylamine precursor, accumulated. Complementation of this strain with the nepAB genes, carried on a plasmid, restored the efflux of nicotine breakdown products. Both NepA and NepB were required for full export activity, indicating that they form a two-component efflux pump. NepAB may function as a metabolic valve by exporting methylamine, the end product of nicotine catabolism, and, in conditions under which it accumulates, the intermediate {gamma}-N-methylaminobutyrate.

Abbreviations: AO, amine oxidase; CCCP, carbonyl cyanide m-chlorophenylhydrazone; 2,6-DHPON, 2,6-dihydroxypseudooxynicotine; 2,6-DHP, 2,6-dihydroxypyridine; 6-HLN, 6-hydroxy-L-nicotine; Kn, kanamycin; LB, lysogeny broth; MABO, {gamma}-N-methylaminobutyrate oxidase; PMF, proton-motive force; PONH, pseudooxynicotine hydrolase; SMR, small multidrug resistance; TPP+, tetraphenylphosphonium






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