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J Bacteriol, May 1998, p. 2599-2608, Vol. 180, No. 10
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Regulation of the Carnitine Pathway in Escherichia coli: Investigation of the cai-fix Divergent Promoter Region

Anne Buchet,* Knut Eichler,dagger and Marie-Andrée Mandrand-Berthelot

Laboratoire de Génétique Moléculaire des Microorganismes et des Interactions Cellulaires, CNRS UMR 5577, Institut National des Sciences Appliquées, F-69621 Villeurbanne Cedex, France

Received 26 November 1997/Accepted 16 March 1998

The divergent structural operons caiTABCDE and fixABCX of Escherichia coli are required for anaerobic carnitine metabolism. Transcriptional monocopy lacZ fusion studies showed that both operons are coexpressed during anaerobic growth in the presence of carnitine, respond to common environmental stimuli (like glucose and nitrate), and are modulated positively by the same general regulators, CRP and FNR, and negatively by H-NS. Overproduction of the CaiF specific regulatory protein mediating the carnitine signal restored induction in an fnr mutant, corresponding to its role as the primary target for anaerobiosis. Transcript analysis identified two divergent transcription start points initiating 289 bp apart. DNase I footprinting revealed three sites with various affinities for the binding of the cAMP-CRP complex inside this regulatory region. Site-directed mutagenesis experiments indicated that previously reported perfect CRP motif 1, centered at -41.5 of the cai transcriptional start site, plays a direct role in the sole cai activation. In contrast, mutation in CRP site 2, positioned at -69.5 of the fix promoter, caused only a threefold reduction in fix expression. Thus, the role of the third CRP site, located at -126.5 of fix, might be to reinforce the action of site 2. A critical 50-bp cis-acting sequence overlapping the fix mRNA start site was found, by deletion analysis, to be necessary for cai transcription. This region is thought to be involved in transduction of the signal mediated by the CaiF regulator.

* Corresponding author. Mailing address: Laboratoire de Génétique Moléculaire des Microorganismes et des Interactions Cellulaires, CNRS UMR 5577, Institut National des Sciences Appliquées, Bâtiment 406, 20, avenue Albert Einstein, F-69621 Villeurbanne Cedex, France. Phone: 33 4 72 43 81 91. Fax: 33 4 72 43 87 14. E-mail: lgmm{at}cismibm.univ-lyon1.fr.

dagger Present address: BASF AG, Zentrale Forschung Feinchemie, D-67056 Ludwigshafen, Germany.

J Bacteriol, May 1998, p. 2599-2608, Vol. 180, No. 10
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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