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Journal of Bacteriology, January 2002, p. 390-399, Vol. 184, No. 2
0021-9193/01/$04.00+0     DOI: 10.1128/JB.184.2.390-399.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Transcriptional Activation of the Rhodobacter sphaeroides Cytochrome c2 Gene P2 Promoter by the Response Regulator PrrA

James C. Comolli, Audrey J. Carl, Christine Hall, and Timothy Donohue*

Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706

Received 17 May 2001/ Accepted 11 October 2001

Anoxygenic photosynthetic growth of Rhodobacter sphaeroides, a member of the {alpha} subclass of the class Proteobacteria, requires the response regulator PrrA. PrrA and the sensor kinase PrrB are part of a two-component signaling pathway that influences a wide range of processes under oxygen-limited conditions. In this work we characterized the pathway of transcription activation by PrrB and PrrA by purifying these proteins, analyzing them in vitro, and characterizing a mutant PrrA protein in vivo and in vitro. When purified, a soluble transmitter domain of PrrB (cPrrB) could autophosphorylate, rapidly transfer phosphate to PrrA, and stimulate dephosphorylation of phospho-PrrA. Unphosphorylated PrrA activated transcription from a target cytochrome c2 gene (cycA) promoter, P2, which contained sequences from -73 to +22 relative to the transcription initiation site. However, phosphorylation of PrrA increased its activity since activation of cycA P2 was enhanced up to 15-fold by treatment with the low-molecular-weight phosphodonor acetyl phosphate. A mutant PrrA protein containing a single amino acid substitution in the presumed phosphoacceptor site (PrrA-D63A) was not phosphorylated in vitro but also was not able to stimulate cycA P2 transcription. PrrA-D63A also had no apparent in vivo activity, demonstrating that aspartate 63 is necessary both for the function of PrrA and for its phosphorylation-dependent activation. The cellular level of wild-type PrrA was negatively autoregulated so that less PrrA was present in the absence of oxygen, conditions in which the activities of many PrrA target genes increase. PrrA-D63A failed to repress expression of the prrA gene under anaerobic conditions, suggesting that this single amino acid change also eliminated PrrA function in vivo.

* Corresponding author. Mailing address: Department of Bacteriology, University of Wisconsin-Madison, Room 312, Fred Hall, Madison, WI 53706. Phone: (608) 262-4663. Fax: (608) 262-9865. E-mail: tdonohue{at}bact.wisc.edu.

Journal of Bacteriology, January 2002, p. 390-399, Vol. 184, No. 2
0021-9193/01/$04.00+0     DOI: 10.1128/JB.184.2.390-399.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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