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Journal of Bacteriology, May 2006, p. 3236-3245, Vol. 188, No. 9
0021-9193/06/$08.00+0     doi:10.1128/JB.188.9.3236-3245.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Mutational Analysis of Intervening Sequences Connecting the Binding Sites for Integration Host Factor, PepA, PurR, and RNA Polymerase in the Control Region of the Escherichia coli carAB Operon, Encoding Carbamoylphosphate Synthase{dagger}

Neel Devroede, Nadine Huysveld, and Daniel Charlier*

Erfelijkheidsleer en Microbiologie, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium

Received 28 December 2005/ Accepted 20 February 2006

Transcription of the carAB operon encoding the unique carbamoylphosphate synthase of Escherichia coli reflects the dual function of carbamoylphosphate in the biosynthesis of arginine and pyrimidine nucleotides. The tandem pair of promoters is regulated by various mechanisms depending on the needs of both pathways and the maintenance of a pyrimidine/purine nucleotide balance. Here we focus on the linker regions that impose the distribution of target sites for DNA-binding proteins involved in pyrimidine- and purine-specific repression of the upstream promoter P1. We introduced deletions and insertions, and combinations thereof, in four linkers connecting the binding sites for integration host factor (IHF), PepA, PurR, and RNA polymerase and studied the importance of phasing and spacing of the targets and the importance of the nucleotide sequence of the linkers. The two PepA binding sites must be properly aligned and separated with respect to each other and to the promoter for both pyrimidine- and purine-mediated repression. Similarly, the phasing and spacing of the IHF and PEPA2 sites are strictly constrained but only for pyrimidine-specific repression. The IHF target is even dispensable for purine-mediated regulation. Thus, a correct localization of PepA within the higher-order nucleoprotein complex is a prerequisite for the establishment of pyrimidine-mediated repression and for the coupling between purine- and pyrimidine-dependent regulation. Our data also suggest the existence of a novel cis-acting pyrimidine-specific regulatory target located around position –60. Finally, the analysis of a P1 derivative devoid of its control region has led to a reappraisal of the effect of excess adenine on P1 and has revealed that P1 has no need for a UP element.

* Corresponding author. Mailing address: Erfelijkheidsleer en Microbiologie (MICR), Pleinlaan 2, B-1050 Brussels, Belgium. Phone: 32 (0)2 629 1342. Fax: 32 (0)2 629 1345. E-mail: dcharlie{at}vub.ac.be.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, May 2006, p. 3236-3245, Vol. 188, No. 9
0021-9193/06/$08.00+0     doi:10.1128/JB.188.9.3236-3245.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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