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J. Biol. Chem., Vol. 279, Issue 28, 28945-28953, July 9, 2004

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Operator Design and Mechanism for CarA Repressor-mediated Down-regulation of the Photoinducible carB Operon in Myxococcus xanthus^*

José Juan López-Rubio, S. Padmanabhan, Jose María Lázaro¶, Margarita Salas¶, Francisco José Murillo||, and Montserrat Elías-Arnanz**

From the Departamento de Genética y Microbiología, Facultad de Biología, Universidad de Murcia, Murcia 30071, and the ^¶Centro de Biología Molecular-Severo Ochoa, Universidad Autónoma, Cantoblanco, Madrid 28049, Spain

The carB operon encodes all except one of the enzymes involved in light-induced carotenogenesis in Myxococcus xanthus. Expression of its promoter (P_B) is repressed in the dark by sequence-specific DNA binding of CarA to a palindrome (pI) located between positions –47 and –64 relative to the transcription start site. This promotes subsequent binding of CarA to additional sites that remain to be defined. CarS, produced in the light, interacts physically with CarA, abrogates CarA-DNA binding, and thereby derepresses P_B. In this study, we delineate the operator design that exists for CarA by precisely mapping out the second operator element. For this, we examined how stepwise deletions and site-directed mutagenesis in the region between the palindrome and the transcription start site affect CarA binding around P_B in vitro and expression of P_B in vivo. These revealed the second operator element to be an imperfect interrupted palindrome (pII) spanning positions –26 to –40. In vitro assays using purified M. xanthus RNA polymerase showed that CarA abolishes P_B-RNA polymerase binding and runoff transcription and that both were restored by CarS, thus rationalizing the observations in vivo. CarA binding to pII (after association with pI) effectively occludes RNA polymerase from P_B and so provides the operative mechanism for the repression of the carB operon by CarA. The bipartite operator design, whereby transcription is blocked by the low affinity CarA-pII binding and is readily restored by CarS, may have evolved to match the needs for a rapid and an effective response to light.

Received for publication, March 29, 2004

^* This work was supported in part by Grants BMC2000-1006 (to F. J. M.), BMC2002-03818 (to M. S.), and BMC2002-00539 and Programa Ramón y Cajal (to S. P.) from the Ministerio de Ciencia y Tecnología, Spain. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a fellowship from Fundación Séneca (Murcia-Spain).

^|| To whom correspondence may be addresssed. Tel.: 34-968-364-951; Fax: 34-968-363-963; E-mail: araujo{at}um.es. ** To whom correspondence may be addressed. Tel.: 34-968-367-134; Fax: 34-968-363-963; E-mail: melias{at}um.es.

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