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Originally published In Press as doi:10.1074/jbc.M211267200 on February 24, 2003

J. Biol. Chem., Vol. 278, Issue 18, 16443-16450, May 2, 2003
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Interactions of the cbbII Promoter-Operator Region with CbbR and RegA (PrrA) Regulators Indicate Distinct Mechanisms to Control Expression of the Two cbb Operons of Rhodobacter sphaeroides*

James M. Dubbs and F. Robert TabitaDagger

From the Department of Microbiology and the Plant Molecular Biology/Biotechnology Program, The Ohio State University, Columbus, Ohio 43210-1292

In a previous study (Dubbs, J. M., Bird, T. H., Bauer, C. E., and Tabita, F. R. (2000) J. Biol. Chem. 275, 19224-19230), it was demonstrated that the regulators CbbR and RegA (PrrA) interacted with both promoter proximal and promoter distal regions of the form I (cbbI) promoter operon specifying genes of the Calvin-Benson-Bassham cycle of Rhodobacter sphaeroides. To determine how these regulators interact with the form II (cbbII) promoter, three cbbFII::lacZ translational fusion plasmids were constructed containing various lengths of sequence 5' to the cbbII operon of R. sphaeroides CAC. Expression of beta -galactosidase was monitored under a variety of growth conditions in both the parental strain and knock-out strains that contain mutations that affect synthesis of CbbR and RegA. The binding sites for both CbbR and RegA were determined by DNase I footprinting. A region of the cbbII promoter from +38 to -227 bp contained a CbbR binding site and conferred low level regulated cbbII expression. The region from -227 to -1025 bp contained six RegA binding sites and conferred enhanced cbbII expression under all growth conditions. Unlike the cbbI operon, the region between -227 and -545 bp that contains one RegA binding site, was responsible for the majority of the observed enhancement. Both RegA and CbbR were required for maximal cbbII expression. Two potentially novel and specific cbbII promoter-binding proteins that did not interact with the cbbI promoter region were detected in crude extracts of R. sphaeroides. These results, combined with the observation that chemoautotrophic expression of the cbbI operon is RegA independent, indicated that the mechanisms controlling cbbI and cbbII operon expression during chemoautotrophic growth are quite different.

* This work was supported by National Institutes of Health Grant GM-45404.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Microbiology, The Ohio State University, 484 West 12th Ave., Columbus, OH 43210-1292. Tel.: 614-292-4297; Fax: 614-292-6337; E-mail: tabita.1@osu.edu.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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