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Applied and Environmental Microbiology, April 2003, p. 2269-2275, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.2269-2275.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Real-Time Reverse Transcription-PCR Analysis of Expression of Halobenzoate and Salicylate Catabolism-Associated Operons in Two Strains of Pseudomonas aeruginosa

M. E. Corbella and A. Puyet*

Departamento de Bioquímica y Biología Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain

Received 12 December 2002/ Accepted 21 January 2003

Pseudomonas aeruginosa JB2 can use 2-chlorobenzoate (2-CBa), 3-CBa, 2,3-dichlorobenzoate (2,3-DCBa), and 2,5-DCBa as sole carbon and energy sources, whereas strain 142 can only grow on 2-CBa and 2,4-DCBa. Both strains, however, harbor the same halobenzoate 1,2-dioxygenase (ohbAB) and chlorocatechol (clcABD) degradation genes necessary for the metabolism of ortho-CBas. In addition, the hybABCD operon, encoding a salicylate 5-hydroxylase, is also found in both strains. The expression of ohbAB, hybABCD, and clcABD operons was measured in cultures grown on different CBas as the sole carbon source and also in glucose-grown cells supplemented with CBas as inducers. A method to standardize real-time reverse transcription-PCR experimental data was used that allows the comparison of semiquantitative mRNA accumulation in different strains and culture conditions. In both strains, the ohb and hyb systems were induced in cells grown on 2-CBa or DCBas, whereas clc was induced only by DCBas. Repression by catabolite was observed both on ohb and clc systems in glucose-grown cells. Chlorocatechol 1,2-dioxygenase activity in JB2 was detected even in clc-repressed conditions, confirming the presence of additional isofunctional genes previously detected in P. aeruginosa 142. Although similar levels of induction of ohbAB were observed in strain JB2 grown on either benzoate, monochlorobenzoates, or DCBas, the ohbAB operon of strain 142 was only strongly induced by growth on 2-CBa and, to a lesser extent, on 2,4-DCBa. This observation suggests that regulation of the ohbAB operon may be different in both strains. The concomitant induction of ohb and hyb by CBas may allow the formation of hybrid halobenzoate dioxygenase(s) composed of Ohb/Hyb dioxygenase subunits and Hyb ferredoxin/ferredoxin reductase components.

* Corresponding author. Mailing address: Departamento de Bioquímica y Biología Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain. Phone: 34-913943827. Fax: 34-913943824. E-mail: apuyet{at}vet.ucm.es.

Applied and Environmental Microbiology, April 2003, p. 2269-2275, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.2269-2275.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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