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Journal of Bacteriology, April 2006, p. 2554-2567, Vol. 188, No. 7
0021-9193/06/$08.00+0     doi:10.1128/JB.188.7.2554-2567.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification of RamA, a Novel LuxR-Type Transcriptional Regulator of Genes Involved in Acetate Metabolism of Corynebacterium glutamicum

Annette Cramer,1 Robert Gerstmeir,1,{ddagger} Steffen Schaffer,2,{dagger} Michael Bott,2 and Bernhard J. Eikmanns1*

Department of Microbiology and Biotechnology, University of Ulm, 89069 Ulm,1 Institute of Biotechnology 1, Research Center Jülich, D-52425 Jülich, Germany2

Received 1 December 2005/ Accepted 17 January 2006

In Corynebacterium glutamicum, the acetate-activating enzymes phosphotransacetylase and acetate kinase and the glyoxylate cycle enzymes isocitrate lyase and malate synthase are coordinately up-regulated in the presence of acetate in the growth medium. This regulation is due to transcriptional control of the respective pta-ack operon and the aceA and aceB genes, brought about at least partly by the action of the negative transcriptional regulator RamB. Using cell extracts of C. glutamicum and employing DNA affinity chromatography, mass spectrometry, and peptide mass fingerprinting, we identified a LuxR-type transcriptional regulator, designated RamA, which binds to the pta-ack and aceA/aceB promoter regions. Inactivation of the ramA gene in the genome of C. glutamicum resulted in mutant RG2. This mutant was unable to grow on acetate as the sole carbon and energy source and, in comparison to the wild type of C. glutamicum, showed very low specific activities of phosphotransacetylase, acetate kinase, isocitrate lyase, and malate synthase, irrespective of the presence of acetate in the medium. Comparative transcriptional cat fusion experiments revealed that this deregulation takes place at the level of transcription. By electrophoretic mobility shift analysis, purified His-tagged RamA protein was shown to bind specifically to the pta-ack and the aceA/aceB promoter regions, and deletion and mutation studies revealed in both regions two binding motifs each consisting of tandem A/C/TG4-6T/C or AC4-5A/G/T stretches separated by four or five arbitrary nucleotides. Our data indicate that RamA represents a novel LuxR-type transcriptional activator of genes involved in acetate metabolism of C. glutamicum.

* Corresponding author. Mailing address: Department of Microbiology and Biotechnology, University of Ulm, 89069 Ulm, Germany. Phone: 49 731 502 2707. Fax: 49 731 502 2710. E-mail: bernhard.eikmanns{at}uni-ulm.de.

{ddagger} Present address: Degussa AG, Project House ProFerm, D-33790 Halle/Westfalen, Germany.

{dagger} Present address: Degussa AG, Project House ProFerm, D-63457 Hanau-Wolfgang, Germany.

Journal of Bacteriology, April 2006, p. 2554-2567, Vol. 188, No. 7
0021-9193/06/$08.00+0     doi:10.1128/JB.188.7.2554-2567.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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