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Applied and Environmental Microbiology, May 2005, p. 2687-2694, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2687-2694.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Polychlorinated Biphenyl Rhizoremediation by Pseudomonas fluorescens F113 Derivatives, Using a Sinorhizobium meliloti nod System To Drive bph Gene Expression

Marta Villacieros,1 Clare Whelan,2 Martina Mackova,3 Jesper Molgaard,4 María Sánchez-Contreras,1 Javier Lloret,1 Daniel Aguirre de Cárcer,1 Roke I. Oruezábal,1 Luis Bolaños,1 Thomas Macek,3 Ulrich Karlson,4 David N. Dowling,2 Marta Martín,1 and Rafael Rivilla1*

Departamento de Biología, Universidad Autónoma de Madrid, 28049 Madrid, Spain,1 Department of Applied Biology & Chemistry, Institute of Technology Carlow, Kilkenny Road, Carlow, Ireland,2 Department of Natural Products, Institute of Organic Chemistry and Biochemistry CAS, and Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, ICT, Prague, Czech Republic,3 Department of Environmental Chemistry & Microbiology, National Environmental Research Institute, P.O. Box 358, Frederiksborgvej 399, 4000 Roskilde, Denmark4

Received 9 September 2004/ Accepted 1 December 2004

Rhizoremediation of organic chemicals requires high-level expression of biodegradation genes in bacterial strains that are excellent rhizosphere colonizers. Pseudomonas fluorescens F113 is a biocontrol strain that was shown to be an excellent colonizer of numerous plant rhizospheres, including alfalfa. Although a derivative of F113 expressing polychlorinated biphenyl (PCB) biodegradation genes (F113pcb) has been reported previously, this strain shows a low level of bph gene expression, limiting its rhizoremediation potential. Here, a high-level expression system was designed from rhizobial nod gene regulatory relays. Nod promoters were tested in strain F113 by using ß-galactosidase transcriptional fusions. This analysis showed that nodbox 4 from Sinorhizobium meliloti has a high level of expression in F113 that is dependent on an intact nodD1 gene. A transcriptional fusion of a nodbox cassette containing the nodD1 gene and nodbox 4 fused to a gfp gene was expressed in the alfalfa rhizosphere. The bph operon from Burkholderia sp. strain LB400 was cloned under the control of the nodbox cassette and was inserted as a single copy into the genome of F113, generating strain F113L::1180. This new genetically modified strain has a high level of BphC activity and grows on biphenyl as a sole carbon and energy source at a growth rate that is more than three times higher than that of F113pcb. Degradation of PCBs 3, 4, 5, 17, and 25 was also much faster in F113L::1180 than in F113pcb. Finally, the modified strain cometabolized PCB congeners present in Delor103 better than strain LB400, the donor of the bph genes used.

* Corresponding author. Mailing address: Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, E-28049 Madrid, Spain. Phone: 34 914978188. Fax: 34 914978344. E-mail address: rafael.rivilla{at}uam.es.

Applied and Environmental Microbiology, May 2005, p. 2687-2694, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2687-2694.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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