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Journal of Bacteriology, June 2005, p. 3678-3686, Vol. 187, No. 11
0021-9193/05/$08.00+0     doi:10.1128/JB.187.11.3678-3686.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Levels and Activity of the Pseudomonas putida Global Regulatory Protein Crc Vary According to Growth Conditions

Ana Ruiz-Manzano, Luis Yuste, and Fernando Rojo*

Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain

Received 12 January 2005/ Accepted 25 February 2005

The global regulatory protein Crc is involved in the repression of several catabolic pathways for sugars, hydrocarbons, and nitrogenated and aromatic compounds in Pseudomonas putida and Pseudomonas aeruginosa when other preferred carbon sources are present in the culture medium (catabolite repression), therefore modulating carbon metabolism. We have analyzed whether the levels or the activity of Crc is regulated. Crc activity was followed by its ability to inhibit the induction by alkanes of the P. putida OCT plasmid alkane degradation pathway when cells grow in a complete medium, where the effect of Crc is very strong. The abundance of crc transcripts and the amounts of Crc protein were higher under repressing conditions than under nonrepressing conditions. The presence of crc on a high-copy-number plasmid considerably increased Crc levels, but this impaired its ability to inhibit the alkane degradation pathway. Crc shows similarity to a family of nucleases that have highly conserved residues at their catalytic sites. Mutation of the corresponding residues in Crc (Asp220 and His246) led to proteins that can inhibit induction of the alkane degradation pathway when present at normal or elevated levels in the cell. Repression by these mutant proteins occurred only under repressing conditions. These results suggest that both the amounts and the activity of Crc are modulated and support previous proposals that Crc may form part of a signal transduction pathway. Furthermore, the activity of the mutant proteins suggests that Crc is not a nuclease.

* Corresponding author. Mailing address: Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain. Phone: (34) 91 585 45 39. Fax: (34) 91 585 45 06. E-mail: frojo{at}cnb.uam.es.

Journal of Bacteriology, June 2005, p. 3678-3686, Vol. 187, No. 11
0021-9193/05/$08.00+0     doi:10.1128/JB.187.11.3678-3686.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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